Bispecific antibodies specifically binding to pd1 and lag3

ABSTRACT

The invention relates to bispecific antibodies comprising a first antigen binding domain that specifically binds to PD1 and a second antigen binding domain that specifically binds to LAG3. The invention further relates to methods of producing these molecules and to methods of using the same.

FIELD OF THE INVENTION

The invention relates to bispecific antibodies comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, in particular tobispecific antibodies further comprising a Fc domain that comprises oneor more amino acid substitution that reduces binding to an Fc receptor,in particular towards Fcγ receptor. The invention further relates tomethods of producing these molecules and to methods of using the same.

BACKGROUND

The importance of the immune system in the protection against cancer isbased on its capacity to detect and destroy abnormal cells. However,some tumor cells are able to escape the immune system by engendering astate of immunosuppression (Zitvogel et al., Nature Reviews Immunology 6(2006), 715-727). T cells have an important role in antiviral andanti-tumour immune responses. Appropriate activation of antigen-specificT cells leads to their clonal expansion and their acquisition ofeffector function, and, in the case of cytotoxic T lymphocytes (CTLs) itenables them to specifically lyse target cells. T cells have been themajor focus of efforts to therapeutically manipulate endogenousantitumour immunity owing to their capacity for the selectiverecognition of peptides derived from proteins in all cellularcompartments; their capacity to directly recognize and killantigen-expressing cells (by CD8⁺ effector T cells; also known ascytotoxic T lymphocytes (CTLs)) and their ability to orchestrate diverseimmune responses (by CD4⁺ helper T cells), which integrates adaptive andinnate effector mechanisms. T cell dysfunction occurs as a result ofprolonged antigen exposure: the T cell loses the ability to proliferatein the presence of the antigen and progressively fails to producecytokines and to lyse target cells1. The dysfunctional T cells have beentermed exhausted T cells and fail to proliferate and exert effectorfunctions such as cytotoxicity and cytokine secretion in response toantigen stimulation. Further studies identified that exhausted T cellsare characterized by sustained expression of the inhibitory moleculePD-1 (programmed cell death protein 1) and that blockade of PD-1 andPD-L1 (PD-1 ligand) interactions can reverse T cell exhaustion andrestore antigen-specific T cell responses in LCMV-infected mice (Barberet al., Nature 439 (2006), 682-687). However, targeting the PD-1-PD-L1pathway alone does not always result in reversal of T cell exhaustion(Gehring et al., Gastroenterology 137 (2009), 682-690), indicating thatother molecules are likely involved in T cell exhaustion (Sakuishi, J.Experimental Med. 207 (2010), 2187-2194).

Lymphocyte activation gene-3 (LAG3 or CD223) was initially discovered inan experiment designed to selectively isolate molecules expressed in anIL-2-dependent NK cell line (Triebel F et al., Cancer Lett. 235 (2006),147-153). LAG3 is a unique transmembrane protein with structuralhomology to CD4 with four extracellular immunoglobulin superfamily-likedomains (D1-D4). The membrane-distal IgG domain contains a short aminoacid sequence, the so-called extra loop that is not found in other IgGsuperfamily proteins. The intracellular domain contains a unique aminoacid sequence (KIEELE, SEQ ID NO:75) that is required for LAG3 to exerta negative effect on T cell function. LAG3 can be cleaved at theconnecting peptide (CP) by metalloproteases to generate a soluble form,which is detectable in serum. Like CD4, the LAG3 protein binds to MHCclass II molecules, however with a higher affinity and at a distinctsite from CD4 (Huard et al. Proc. Natl. Acad. Sci. USA 94 (1997),5744-5749). LAG3 is expressed by T cells, B cells, NK cells andplasmacytoid dendritic cells (pDCs) and is upregulated following T cellactivation. It modulates T cell function as well as T cell homeostasis.Subsets of conventional T cells that are anergic or display impairedfunctions express LAG3. LAG3⁺ T cells are enriched at tumor sites andduring chronic viral infections (Sierro et al Expert Opin. Ther. Targets15 (2011), 91-101). It has been shown that LAG3 plays a role in CD8 Tcell exhaustion (Blackburn et al. Nature Immunol. 10 (2009), 29-37).Thus, there is a need for antibodies that antagonize the activity ofLAG3 and can be used to generate and restore immune response to tumors.

Monoclonal antibodies to LAG3 have been described, for example, in WO2004/078928 wherein a composition comprising antibodies specificallybinding to CD223 and an anti-cancer vaccine is claimed. WO 2010/019570discloses human antibodies that bind LAG3, for example the antibodies25F7 and 26H10. US 2011/070238 relates to a cytotoxic anti-LAG3 antibodyuseful in the treatment or prevention of organ transplant rejection andautoimmune disease. WO 2014/008218 describes LAG3 antibodies withoptimized functional properties (i.e. reduced deamidation sites)compared to antibody 25F7. Furthermore, LAG3 antibodies are disclosed inWO 2015/138920 (for example BAP050), WO 2014/140180, WO 2015/116539, WO2016/028672, WO 2016/126858, WO 2016/200782 and WO 2017/015560.

Programmed cell death protein 1 (PD-1 or CD279) is an inhibitory memberof the CD28 family of receptors, that also includes CD28, CTLA-4, ICOSand BTLA. PD-1 is a cell surface receptor and is expressed on activatedB cells, T cells, and myeloid cells (Okazaki et al (2002) Curr. Opin.Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8). Thestructure of PD-1 is a monomeric type 1 transmembrane protein,consisting of one immunoglobulin variable-like extracellular domain anda cytoplasmic domain containing an immunoreceptor tyrosine-basedinhibitory motif (ITIM) and an immunoreceptor tyrosine-based switchmotif (ITSM). Activated T cells transiently express PD1, but sustainedhyperexpression of PD1 and its ligand PDL1 promote immune exhaustion,leading to persistence of viral infections, tumor evasion, increasedinfections and mortality. PD1 expression is induced by antigenrecognition via the T-cell receptor and its expression is maintainedprimarily through continuous T-cell receptor signaling. After prolongedantigen exposure, the PD1 locus fails to be remethylated, which promotescontinuous hyperexpression. Blocking the PD1 pathway can restore theexhausted T-cell functionality in cancer and chronic viral infections(Sheridan, Nature Biotechnology 30 (2012), 729-730). Monoclonalantibodies to PD-1 have been described, for example, in WO 2003/042402,WO 2004/004771, WO 2004/056875, WO 2004/072286, WO 2004/087196, WO2006/121168, WO 2006/133396, WO 2007/005874, WO 2008/083174, WO2008/156712, WO 2009/024531, WO 2009/014708, WO 2009/101611, WO2009/114335, WO 2009/154335, WO 2010/027828, WO 2010/027423, WO2010/029434, WO 2010/029435, WO 2010/036959, WO 2010/063011, WO2010/089411, WO 2011/066342, WO 2011/110604, WO 2011/110621, WO2012/145493, WO 2013/014668, WO 2014/179664, and WO 2015/112900.

Bispecific Fc diabodies having immunoreactivity with PD1 and LAG3 foruse in the treatment of cancer or a disease associated with a pathogensuch as a bacterium, a fungus or a virus are described in WO2015/200119. However, there is a need of providing new bispecificantibodies that not only simultaneously bind to PD1 and LAG3 and thusselectively target cells expressing both PD1 and LAG3, but that alsoavoid blocking of LAG3 on other cells given the broad expression patternof LAG3. The bispecific antibodies of the present invention do not onlyeffectively block PD1 and LAG3 on T cells overexpressing both PD1 andLAG3, they are very selective for these cells and thereby side effectsby administering highly active LAG3 antibodies may be avoided.

SUMMARY OF THE INVENTION

The present invention relates to bispecific antibodies comprising atleast one antigen binding domain that specifically binds to programmedcell death protein 1 (PD1) and at least one second antigen bindingdomain that specifically binds to Lymphocyte activation gene-3 (LAG3).These bispecific antibodies are advantageous as they provide betterselectivity and, potentially, efficacy than anti-PD1 and anti-LAG3combination strategies. They are further characterized in that show areduced sink effect (as shown by reduced internalization by T cells),they preferentially bind to conventional T cells as to Tregs and areable to rescue T cell effector functions from Treg suppression, theyshow increased tumor-specific T cell effector functions and increasedtumor eradication in vivo.

In one aspect, the invention provides a bispecific antibody comprising afirst antigen binding domain that specifically binds to programmed celldeath protein 1 (PD1) and a second antigen binding domain thatspecifically binds to Lymphocyte activation gene-3 (LAG3), wherein

-   -   said first antigen binding domain specifically binding to PD1        comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:            1,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:2, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:3; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:4;        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:5, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:6.

In particular, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to programmed cell deathprotein 1 (PD1) and a second antigen binding domain that specificallybinds to Lymphocyte activation gene-3 (LAG3), wherein the bispecificantibody comprises a Fc domain that is an IgG, particularly an IgG1 Fcdomain or an IgG4 Fc domain and wherein the Fc domain comprises one ormore amino acid substitution that reduces binding to an Fc receptor, inparticular towards Fcγ receptor.

In one aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thesecond antigen binding domain that specifically binds to LAG3 comprises

-   -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:            14,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:            15, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:            16; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:            17.        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:            18, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO: 19; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:22,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:23, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:24; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:25,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:26, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:27; or    -   (c) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:30,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:31, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:32; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:33,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:34, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:35; or    -   (d) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:38,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:39, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:40; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:41,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:42, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:43; or    -   (e) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:46.        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:47, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:48; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:49,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:50, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:51.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thefirst antigen-binding domain specifically binding to PD1 comprises

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        7 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 8, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 10, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 11, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 12, or    -   (e) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 13.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, therein thefirst antigen-binding domain specifically binding to PD1 comprises a VHdomain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein the second antigen-binding domain specificallybinding to LAG3 comprises

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        20 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 21, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        28 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 29, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        36 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 37, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        44 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 45, or    -   (e) a VH domain comprising the amino acid sequence of SEQ ID NO:        52 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 53.

In an additional aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein the second antigen-binding domain specificallybinding to LAG3 comprises

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        54 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 55, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        62 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 63, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        64 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 65, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        66 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 67.

Furthermore, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein

-   -   the first antigen binding domain specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 9 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 10,    -   and the second antigen binding domain specifically binding to        LAG3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 20 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 21 or a VH domain comprising the amino acid        sequence of SEQ ID NO: 52 and a VL domain comprising the amino        acid sequence of SEQ ID NO: 53.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein

-   -   the first antigen binding domain specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 9 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 10,    -   and the second antigen binding domain specifically binding to        LAG3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 20 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 21.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein

-   -   the first antigen binding domain specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 9 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 10,    -   and the second antigen binding domain specifically binding to        LAG3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 56 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 57.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein the bispecific antibody is a humanized orchimeric antibody. In particular, the bispecific antibody is a humanizedantibody.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein the bispecific antibody comprises an Fc domain ofhuman IgG1 subclass with the amino acid mutations L234A, L235A and P329G(numbering according to Kabat EU index).

Furthermore, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein the bispecific antibody comprises an Fc domaincomprising a modification promoting the association of the first andsecond subunit of the Fc domain. In one aspect, provided is a bispecificantibody, wherein the first subunit of the Fc domain comprises knobs andthe second subunit of the Fc domain comprises holes according to theknobs into holes method. In particular, the first subunit of the Fcdomain comprises the amino acid substitutions S354C and T366W (EUnumbering) and the second subunit of the Fc domain comprises the aminoacid substitutions Y349C, T366S and Y407V (numbering according to KabatEU index).

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, a first Fab fragmentcomprising the antigen binding domain that specifically binds to PD1 anda second Fab fragment comprising the antigen binding domain thatspecifically binds to LAG3.

In one aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein in oneof the Fab fragments the variable domains VL and VH are replaced by eachother so that the VH domain is part of the light chain and the VL domainis part of the heavy chain. Particularly, provided is bispecificantibody, wherein in the first Fab fragment comprising the antigenbinding domain that specifically binds to PD1 the variable domains VLand VH are replaced by each other.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises a Fab fragment wherein in the constantdomain CL the amino acid at position 124 is substituted independently bylysine (K), arginine (R) or histidine (H) (numbering according to KabatEU Index), and in the constant domain CH1 the amino acids at positions147 and 213 are substituted independently by glutamic acid (E) oraspartic acid (D) (numbering according to Kabat EU index). Particularly,provided is bispecific antibody, wherein in the second Fab fragmentcomprising the antigen binding domain that specifically binds to LAG3the constant domain CL the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat EU Index), and in the constant domain CH1 the aminoacids at positions 147 and 213 are substituted independently by glutamicacid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, comprising

-   -   (a) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO: 96,        a first light chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 98,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            97, and a second light chain comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO:99, or    -   (b) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO: 96,        a first light chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 98,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            100, and a second light chain comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO: 101, or    -   (c) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        102, a first light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        104,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            103, and a second light chain comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO: 105, or    -   (d) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        106, a first light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        107,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            103, and a second light chain comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO: 105.

More particularly, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, comprising anamino acid sequence with at least 95% sequence identity to the sequenceof SEQ ID NO: 96, a first light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 98, asecond heavy chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 100, and a second lightchain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 101.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein the bispecific antibody comprises a Fab fragmentcomprising the antigen binding domain that specifically binds to LAG3which is fused to the C-terminus of the Fc domain.

In particular, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, comprising anamino acid sequence with at least 95% sequence identity to the sequenceof SEQ ID NO: 96, a first light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 98, asecond heavy chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 144, and a second lightchain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 101.

In a further aspect provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein the bispecific antibody comprises a third Fabfragment comprising an antigen binding domain that specifically binds toLAG3. In one aspect, provided is a bispecific antibody, wherein the twoFab fragments comprising each an antigen binding domain thatspecifically binds to LAG3 are identical.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein the Fab fragment comprising the antigen bindingdomain that specifically binds to PD1 is fused via a peptide linker tothe C-terminus of one of the heavy chains.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, comprising

-   -   (a) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        118, a first light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        115,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            119, and two second light chains comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO: 101, or    -   (b) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        120, a first light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        115,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            121, and two second light chains comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO:99, or    -   (c) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        122, a first light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        115,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            103, and two second light chains comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO: 105.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein one of the Fab fragments comprising the antigenbinding domain that specifically binds to LAG3 is fused via a peptidelinker to the C-terminus of one of the heavy chains.

In a particular aspect provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, comprising anamino acid sequence with at least 95% sequence identity to the sequenceof SEQ ID NO: 96, a first light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 98, asecond heavy chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 145, and two secondlight chains comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 101.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein the bispecific antibody comprises a fourth Fabfragment comprising an antigen binding domain that specifically binds toPD1. In one aspect, provided is a bispecific antibody, wherein the twoFab fragments comprising each an antigen binding domain thatspecifically binds to PD1 are identical.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein before, wherein the two Fab fragments comprising each an antigenbinding domain that specifically binds to PD1 are each fused via apeptide linker to the C-terminus to one of the heavy chains,respectively.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, comprising

-   -   (a) two heavy chains comprising each an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        114, two first light chains comprising each an amino acid        sequence with at least 95% sequence identity to the sequence of        SEQ ID NO: 115, and two second light chains comprising each an        amino acid sequence with at least 95% sequence identity to the        sequence of SEQ ID NO: 101, or    -   (b) two heavy chains comprising each an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        116, two first light chains comprising each an amino acid        sequence with at least 95% sequence identity to the sequence of        SEQ ID NO: 115, and two second light chains comprising each an        amino acid sequence with at least 95% sequence identity to the        sequence of SEQ ID NO:99, or    -   (c) two heavy chains comprising each an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        117, two first light chains comprising each an amino acid        sequence with at least 95% sequence identity to the sequence of        SEQ ID NO: 115, and two second light chains comprising an amino        acid sequence with at least 95% sequence identity to the        sequence of SEQ ID NO: 105.

In yet another aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, two Fab fragments comprisingeach an antigen binding domain that specifically binds to LAG3 and asingle chain Fab (scFab) comprising the antigen binding domain thatspecifically binds to PD1. In particular, the scFab comprising anantigen binding domain that specifically binds to PD1 is fused via apeptide linker to the C-terminus to one of the heavy chains.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, comprising

-   (a) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 123, a    second heavy chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 119, and two    light chains comprising each an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 101, or-   (b) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 124, a    second heavy chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 121, and two    light chains comprising each an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO:99, or-   (c) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 125, a    second heavy chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 103, and a    second light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 105.

In yet another aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, two Fab fragments comprisingeach an antigen binding domain that specifically binds to LAG3 and a VHand VL domain comprising the antigen binding domain that specificallybinds to PD1. In one aspect, the VH domain of the antigen binding domainthat specifically binds to PD1 is fused via a peptide linker to theC-terminus of one of the heavy chains and the VL domain of the antigenbinding domain that specifically binds to PD1 is fused via a peptidelinker to the C-terminus of the other one of the heavy chains. In aparticular aspect, provided is a bispecific antibody, comprising a firstheavy chain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 126, a second heavy chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 127, and two light chains comprising each anamino acid sequence with at least 95% sequence identity to the sequenceof SEQ ID NO: 109.

According to another aspect of the invention, there is provided apolynucleotide encoding the bispecific antibody as described hereinbefore. The invention further provides a vector, particularly anexpression vector, comprising a polynucleotide of the invention and aprokaryotic or eukaryotic host cell comprising the polynucleotide or thevector of the invention. In some embodiments the host cell is aeukaryotic cell, particularly a mammalian cell.

In another aspect, provided is a method for producing bispecificantibody comprising a first antigen binding domain that specificallybinds to PD1 and a second antigen binding domain that specifically bindsto LAG3 as described herein, comprising the steps of a) transforming ahost cell with vectors comprising polynucleotides encoding saidbispecific antibody, b) culturing the host cell according underconditions suitable for the expression of the bispecific antibody and c)recovering the bispecific antibody from the culture. The invention alsoencompasses a bispecific antibody produced by the method of theinvention.

The invention further provides a pharmaceutical composition comprising abispecific antibody comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 as described herein, and at least onepharmaceutically acceptable excipient.

Also encompassed by the invention is the bispecific antibody comprisinga first antigen binding domain that specifically binds to PD1 and asecond antigen binding domain that specifically binds to LAG3 asdescribed herein, or the pharmaceutical composition comprising thebispecific antibody, for use as a medicament.

In another aspect, the invention provides a bispecific antibodycomprising a bispecific antibody comprising a first antigen bindingdomain that specifically binds to PD1 and a second antigen bindingdomain that specifically binds to LAG3 as described herein, or thepharmaceutical composition comprising the bispecific antibody, for use

-   i) in the modulation of immune responses, such as restoring T cell    activity,-   ii) in stimulating an immune response or function,-   iii) in the treatment of infections,-   iv) in the treatment of cancer,-   v) in delaying progression of cancer,-   vi) in prolonging the survival of a patient suffering from cancer.

In one aspect, provided is the bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein, or the pharmaceutical composition comprising the bispecificantibody, for use in the treatment of a disease in an individual in needthereof. In a specific aspect, the invention provides a bispecificantibody comprising a first antigen binding domain that specificallybinds to PD1 and a second antigen binding domain that specifically bindsto LAG3, or the pharmaceutical composition comprising the bispecificantibody, for use in the treatment of cancer. In a further specificaspect, a bispecific antibody comprising a first antigen binding domainthat specifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3, or the pharmaceutical composition comprisingthe bispecific antibody, for use in the modulation of immune responsesis provided. In another aspect, a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, or apharmaceutical composition comprising the bispecific antibody for use inthe treatment of a chronic viral infection is provided.

The invention also provides a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein, or a pharmaceutical composition comprising the bispecificantibody for use in the prevention or treatment of cancer, wherein thebispecific antibody is administered in combination with achemotherapeutic agent, radiation and/or other agents for use in cancerimmunotherapy. In a particular aspect, provided is bispecific antibodycomprising a first antigen binding domain that specifically binds to PD1and a second antigen binding domain that specifically binds to LAG3 asdescribed herein, or a pharmaceutical composition comprising thebispecific antibody for use in the prevention or treatment of cancer,wherein the bispecific antibody is administered in combination with ananti-CEA/anti-CD3 bispecific antibody.

Also provided is the use of the bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein for the manufacture of a medicament for the treatment of adisease in an individual in need thereof, in particular for themanufacture of a medicament for the treatment of cancer, as well as amethod of treating a disease in an individual, comprising administeringto said individual a therapeutically effective amount of a compositioncomprising a bispecific antibody comprising a first antigen bindingdomain that specifically binds to PD1 and a second antigen bindingdomain that specifically binds to LAG3 as described herein in apharmaceutically acceptable form. In a specific aspect, the disease iscancer. In another specific aspect, the disease is a chronic viralinfection. In another aspect, a method of modulating of immune responsesin an individual, comprising administering to said individual atherapeutically effective amount of a composition comprising thebispecific antibody comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 as described herein in a pharmaceuticallyacceptable form is provided. In any of the above aspects the individualis preferably a mammal, particularly a human.

The invention also provides a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein, or a pharmaceutical composition comprising the bispecificantibody for use in the prevention or treatment of cancer, wherein thebispecific antibody is administered in combination with achemotherapeutic agent, radiation and/or other agents for use in cancerimmunotherapy.

Furthermore, provided is a method of inhibiting the growth of tumorcells in an individual comprising administering to the individual aneffective amount of a bispecific antibody comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 as described herein toinhibit the growth of the tumor cells. The individual is preferably amammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1I: Schematic illustration of the different formats of thebispecific anti-PD1/anti-LAG3 antibodies described herein. FIG. 1A showsthe bispecific 1+1 format, wherein the PD1 binding domain comprises acrossFab (with VH/VL domain exchange) and the LAG3 binding domaincomprises CH1 and CK domains with amino acid mutations to supportcorrect pairing (“charged variants”). The Fc part comprises the knobinto hole mutations (illustrated by the black arrow) and the amino acidmutations L234A, L235A and P329G almost completely abolishing Fcγreceptor binding of the human IgG1 Fc domain (illustrated by the whitearea). FIG. 1B shows a 2+1 format with two anti-LAG3 binding Fab domainscomprising mutations in CHUCK and a PD1 binding Fab domain fused at theC-terminus of one heavy chain. FIG. 1C shows a similar 2+1 format withtwo anti-LAG3 binding FAB domains comprising mutations in CH1/CK, but aPD1 binding single chain scFab domain fused at the C-terminus of oneheavy chain. In FIG. 1D is shown a 2+1 format with two anti-LAG3 bindingFab domains and a PD1 binding VH and VL fused each to one of theC-termini of the heavy chains. FIG. 1E shows a construct similar to saidof FIG. 1D, however with an engineered disulfide bond between VH and VLand in FIG. 1F a variant with a Furin site is shown. FIG. 1G shows thebispecific 2+2 format with two anti-LAG3 binding Fab domains comprisingmutations in CH1/CK and two PD1 binding crossFab domains fused at theC-terminus of each heavy chain. In FIG. 1H a bispecific 1+1 format isshown (called “trans”), wherein the PD1 binding domain comprises acrossFab (with VH/VL domain exchange) and the LAG3 binding domain isfused with its VH domain at the C-terminus of the Fc hole chain. TheLag3 domain comprises CH1 and CK domains with amino acid mutations tosupport correct pairing (“charged variants”). FIG. 1I shows a 2+1 transformat, wherein the PD1 binding domain comprises a crossFab (with VH/VLdomain exchange) and one LAG3 binding domain comprising CH1 and CKdomains with amino acid mutations to support correct pairing (“chargedvariants”) and a second LAG3 binding domain is fused with its VH domainat the C-terminus of the Fc hole chain.

FIGS. 2A and 2B: Effect of aLAG-3 antibodies on cytotoxic Granzyme Brelease and IL-2 secretion by human CD4 T cells cocultured withallogeneic mature dendritic cells. In FIG. 2A the effect of aLAG3antibodies as described herein on Granzyme B secretion and in FIG. 2Bthe effect of aLAG3 antibodies on IL-2 secretion is shown.

FIG. 3: Effect of aLAG3 antibodies in combination with aPD1 antibody(0376) on cytotoxic Granzyme B release by human CD4 T cells coculturedwith a B cell-lymphoblatoid cell line (ARH77). Shown is a comparison ofdifferent aLAG3 antibodies in combination with aPD1 antibody (0376) andwith aPD1 antibody (0376) alone.

FIGS. 4A and 4B: Effect of aLAG3 antibodies in combination with aPD1antibody (0376) on Treg suppression of Granzyme B and IFN-γ release byhuman CD4 T cells cocultured with irradiated allogeneic PBMCs. FIG. 4Ashows the Granzyme B release in comparison with aPD1 (0376) alone andFIG. 4B shows the IFN-γ release in comparison with aPD1 (0376) alone.

FIGS. 5A-5D: Simultaneous binding and receptor dimerization caused bybinding of bispecific anti-PD1/anti-LAG3 antibodies to recombinantPD1⁺Lag3⁺ cells. Plotted is the chemoluminescence (measured in RU)against the antibody concentration. FIG. 5A and FIG. 5B show acomparison of bispecific anti-PD1/anti-LAG3 antibodies and monospecificanti-LAG3 antibodies. Only the bispecific formats were able to inducechemoluminescence. A competition experiment is shown in FIG. 5C. If thesame bispecific antibody was provided in the presence of either an aLAG3antibody (0156, MDX25F7) or anti-PD1 antibody (0376), the signal waseither almost inhibited (for PD1 competition) or at least significantlyreduced (Lag3). A further competition experiment is shown in FIG. 5D.Competition of the bispecific anti-PD1/anti-LAG3 antibody with the sameanti-PD1 antibody (0376) and also recombinant LAG3:Fc protein (0160)almost abolished the signal, whereas presence of the same aLAG3 binder(0156) only led to partial inhibition and two further anti-LAG3antibodies 0414 and 0416 did not modulate the signal significantly.

FIGS. 6A-6D: Comparison of the simultaneous binding of bispecificanti-PD1/anti-LAG3 antibodies in different formats (1+1 vs. 2+1) andwith different aLAG3 binders. FIG. 6A shows the binding curve forconstruct 0799 (anti-PD1(0376)/anti-LAG3(0416) in 1+1 format). Thebinding curve for construct 8311 (anti-PD1(0376)/anti-LAG3(0416) in 1+2format) is shown in FIG. 6B. FIG. 6C shows the binding curve forconstruct 0927 (anti-PD1(0376)/anti-LAG3(0414) in 1+1 format). Thebinding curve for construct 8310 (anti-PD1(0376)/anti-LAG3(0414) in 1+2format) is shown in FIG. 6D.

FIGS. 7A-7F: Comparison of the simultaneous binding of bispecificanti-PD1/anti-LAG3 antibodies in different formats (2+1 vs. 2+2) andwith different aLAG3 binders. FIG. 7A shows the binding curve forconstruct 8310 (anti-PD1(0376)/anti-LAG3(0414) in 1+2 format). Thebinding curve for construct 8970 (anti-PD1 (0376)/anti-LAG3(0414) in 2+2format) is shown in FIG. 7B. FIG. 7C shows the binding curve forconstruct 8311 (anti-PD1(0376)/anti-LAG3(0416) in 1+2 format). Thebinding curve for construct 8984 (anti-PD1(0376)/anti-LAG3(0416) in 2+2format) is shown in FIG. 7D. The binding curves for constructs 0725(anti-PD1 (0376)/anti-LAG3(0414) in trans 1+1 format) and 0750(anti-PD1(0376)/anti-LAG3(0414) in trans 1+2 format) are shown in FIG.7E in comparison to the binding curve of construct 0927 (anti-PD1(0376)/anti-LAG3(0414) in 1+1 format). These 3 constructs were alsocompared in the commercially available PD1/LAG3 combo Reporter assay andthe corresponding binding curves are shown in FIG. 7F.

FIGS. 8A and 8B: Internalization of bispecific anti-PD1/anti-LAG3antibodies in different formats ̂ and parental anti-LAG3 antibody after3 hours from the addition to administration to activated T cells asmeasured with flow cytometry. FIG. 8A shows the representative histogramof the experiment, the percentage of internalization for the differentformats is shown in FIG. 8B.

FIGS. 9A and 9B: Analysis over time shows higher membrane localizationof the 1+1 format of the bispecific anti-PD1/anti-LAG3 antibody (0927)when compared to the other formats which show a higher degree ofinternalization. FIG. 9A shows the fluorescent images as detected byconfocal microscopy after 15 minutes, 1 hour and 3 hours. The activatedCD4 cells are shown as black balls. The fluorescent images for a TIM3antibody are shown as an example for strong internalization. Aquantitatve analysis of the images is shown in FIG. 9B.

FIGS. 10A-10D: Binding to conventional T cells versus Tregs. FIGS. 10Ato 10C show data from one representative donor showing the binding toconventional T cells (black curve) and Tregs (grey area). The binding ofan anti-LAG3 antibody 0414 (hu IgG1 PGLALA) is shown in FIG. 10A, FIG.10B and FIG. 10C show the binding of anti-PD1 antibody 0376 andbispecific anti-PD1/anti-LAG3 antibody (0927), respectively. In FIG. 10Dthe Delta of the geometric fluorescent mean intensity of a givenmolecule bound on conventional T cells versus the one on Tregs withinthe same sample are shown. Results (Median) are from 3 independentexperiments with 3 different donors.

FIG. 11: PD1 and Treg co-blockade rescues tconv effector functions fromTreg suppression. Shown is the percentage of suppression by Tregs ofgranzyme B secreted by Tconv after 5 days of coculture. Results (Median)are from 10 independent experiments with 10 different donors. P wascalculated using two-way ANOVA.

FIG. 12: Effect of PD-1 and LAG-3 blockade on Granzyme B and IFN-γsecretion by CD4 T cells from melanoma patient PBMCs after recall withimmunogenic melanoma-antigen peptide pools. FIG. 12 shows a comparisonof the effect on Granzyme B and IFN-γ release caused by anti-PD1(0376)alone, the combination of anti-PD1(0376) with aLAG3(0414) and thebispecific antibody 0927 (anti-PD1(0376)/anti-LAG3(0414) in 1+1 format).Shown is the fold increase in granzyme B and IFNγ production relative topeptide-pool stimulated CD4 T cells from 12 melanoma patient PBMCs.

FIG. 13: Effect of aPD1/aLAG3 bispecific antibodies on cytotoxicGranzyme B release by human CD4 T cells cocultured with a Bcell-lymphoblatoid cell line (ARH77). Different bispecificanti-PD1/anti-LAG3 antibodies as described herein are compared withantibodies used in standard of care or clinical trials.

FIG. 14: Efficacy study in humanized mice challenged with pancreaticadenocarcinoma, BxPC3. In combination with CEACAM CD3 TCB, only theaPD1/aLAG3 bispecific antibody provided a statistical significant tumorprotection when compared to conventional PD1 antibodies. Shown are thetumor growth curves in humanized mice challenged subcutaneously withBxPC3 cells and treated with the indicated molecules in combination withCEACAM5-TCB.

FIG. 15A-15F: The measurements of tumor volumes (mm³+/−SEM), over aperiod of 47 days, are shown for each individual animal showing thehomogeneity of group anti-tumor response. The tumor growth curves areshown for the vehicle group in FIG. 15A, for CEACAM5 CD3 TCB alone (2.5mg/kg) in FIG. 15B, for the combination of CEACAM5 CD3 TCB withNivolumab (1.5 mg/kg) in FIG. 15C, for the combination of CEACAM5 CD3TCB with Pembrolizumab (1.5 mg/kg) in FIG. 15D, for the combination ofCEACAM5 CD3 TCB with PD1/LAG3 0927 in FIG. 15E (1.5 mg/kg) and in FIG.15F (3 mg/kg bispecific antibody).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as generally used in the art to which thisinvention belongs. For purposes of interpreting this specification, thefollowing definitions will apply and whenever appropriate, terms used inthe singular will also include the plural and vice versa.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are antibodies,antibody fragments and scaffold antigen binding proteins.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, monospecific and multispecificantibodies (e.g., bispecific antibodies), and antibody fragments so longas they exhibit the desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g. containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen. The term “bispecific” means that the antibody is ableto specifically bind to at least two distinct antigenic determinants,for example two binding sites each formed by a pair of an antibody heavychain variable domain (VH) and an antibody light chain variable domain(VL) binding to different antigens or to different epitopes on the sameantigen. Such a bispecific antibody is an 1+1 format. Other bispecificantibody formats are 2+1 formats (comprising two binding sites for afirst antigen or epitope and one binding site for a second antigen orepitope) or 2+2 formats (comprising two binding sites for a firstantigen or epitope and two binding sites for a second antigen orepitope). Typically, a bispecific antibody comprises two antigen bindingsites, each of which is specific for a different antigenic determinant.

The term “valent” as used within the current application denotes thepresence of a specified number of binding domains in an antigen bindingmolecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent”denote the presence of two binding domain, four binding domains, and sixbinding domains, respectively, in an antigen binding molecule. Thebispecific antibodies according to the invention are at least “bivalent”and may be “trivalent” or “multivalent” (e.g. “tetravalent” or“hexavalent”). In a particular aspect, the antibodies of the presentinvention have two or more binding sites and are bispecific. That is,the antibodies may be bispecific even in cases where there are more thantwo binding sites (i.e. that the antibody is trivalent or multivalent).

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG-classantibodies are heterotetrameric glycoproteins of about 150,000 daltons,composed of two light chains and two heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by alight chain constant domain (CL), also called a light chain constantregion. The heavy chain of an antibody may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2),γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies, triabodies, tetrabodies, cross-Fab fragments; linearantibodies; single-chain antibody molecules (e.g. scFv); multispecificantibodies formed from antibody fragments and single domain antibodies.For a review of certain antibody fragments, see Hudson et al., Nat Med9, 129-134 (2003). For a review of scFv fragments, see e.g. Plückthun,in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg andMoore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion ofFab and F(ab′)2 fragments comprising salvage receptor binding epitoperesidues and having increased in vivo half-life, see U.S. Pat. No.5,869,046. Diabodies are antibody fragments with two antigen bindingdomains that may be bivalent or bispecific, see, for example, EP404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); andHollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat Med9, 129-134 (2003). Single-domain antibodies are antibody fragmentscomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1).In addition, antibody fragments comprise single chain polypeptideshaving the characteristics of a VH domain, namely being able to assembletogether with a VL domain, or of a VL domain, namely being able toassemble together with a VH domain to a functional antigen binding siteand thereby providing the antigen binding property of full lengthantibodies.

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

Papain digestion of intact antibodies produces two identicalantigen-binding fragments, called “Fab” fragments containing each theheavy- and light-chain variable domains and also the constant domain ofthe light chain and the first constant domain (CH1) of the heavy chain.As used herein, Thus, the term “Fab fragment” refers to an antibodyfragment comprising a light chain fragment comprising a VL domain and aconstant domain of a light chain (CL), and a VH domain and a firstconstant domain (CH1) of a heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteins from theantibody hinge region. Fab′-SH are Fab′ fragments wherein the cysteineresidue(s) of the constant domains bear a free thiol group. Pepsintreatment yields an F(ab′)₂ fragment that has two antigen-combiningsites (two Fab fragments) and a part of the Fc region.

The term “cross-Fab fragment” or “xFab fragment” or “crossover Fabfragment” refers to a Fab fragment, wherein either the variable regionsor the constant regions of the heavy and light chain are exchanged. Twodifferent chain compositions of a crossover Fab molecule are possibleand comprised in the bispecific antibodies of the invention: On the onehand, the variable regions of the Fab heavy and light chain areexchanged, i.e. the crossover Fab molecule comprises a peptide chaincomposed of the light chain variable region (VL) and the heavy chainconstant region (CH1), and a peptide chain composed of the heavy chainvariable region (VH) and the light chain constant region (CL). Thiscrossover Fab molecule is also referred to as CrossFab_((VLVH)). On theother hand, when the constant regions of the Fab heavy and light chainare exchanged, the crossover Fab molecule comprises a peptide chaincomposed of the heavy chain variable region (VH) and the light chainconstant region (CL), and a peptide chain composed of the light chainvariable region (VL) and the heavy chain constant region (CH1). Thiscrossover Fab molecule is also referred to as CrossFab_((CLCH1)).

A “single chain Fab fragment” or “scFab” is a polypeptide consisting ofan antibody heavy chain variable domain (VH), an antibody constantdomain 1 (CH1), an antibody light chain variable domain (VL), anantibody light chain constant domain (CL) and a linker, wherein saidantibody domains and said linker have one of the following orders inN-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b)VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL;and wherein said linker is a polypeptide of at least 30 amino acids,preferably between 32 and 50 amino acids. Said single chain Fabfragments are stabilized via the natural disulfide bond between the CLdomain and the CH1 domain. In addition, these single chain Fab moleculesmight be further stabilized by generation of interchain disulfide bondsvia insertion of cysteine residues (e.g. position 44 in the variableheavy chain and position 100 in the variable light chain according toKabat numbering).

A “crossover single chain Fab fragment” or “x-scFab” is a is apolypeptide consisting of an antibody heavy chain variable domain (VH),an antibody constant domain 1 (CH1), an antibody light chain variabledomain (VL), an antibody light chain constant domain (CL) and a linker,wherein said antibody domains and said linker have one of the followingorders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 andb) VL-CH1-linker-VH-CL; wherein VH and VL form together an antigenbinding domain which binds specifically to an antigen and wherein saidlinker is a polypeptide of at least 30 amino acids. In addition, thesex-scFab molecules might be further stabilized by generation ofinterchain disulfide bonds via insertion of cysteine residues (e.g.position 44 in the variable heavy chain and position 100 in the variablelight chain according to Kabat numbering).

A “single-chain variable fragment (scFv)” is a fusion protein of thevariable regions of the heavy (V_(H)) and light chains (V_(L)) of anantibody, connected with a short linker peptide of ten to about 25 aminoacids. The linker is usually rich in glycine for flexibility, as well asserine or threonine for solubility, and can either connect theN-terminus of the V_(H) with the C-terminus of the V_(L), or vice versa.This protein retains the specificity of the original antibody, despiteremoval of the constant regions and the introduction of the linker. scFvantibodies are, e.g. described in Houston, J. S., Methods in Enzymol.203 (1991) 46-96). In addition, antibody fragments comprise single chainpolypeptides having the characteristics of a VH domain, namely beingable to assemble together with a VL domain, or of a VL domain, namelybeing able to assemble together with a VH domain to a functional antigenbinding site and thereby providing the antigen binding property of fulllength antibodies.

“Scaffold antigen binding proteins” are known in the art, for example,fibronectin and designed ankyrin repeat proteins (DARPins) have beenused as alternative scaffolds for antigen-binding domains, see, e.g.,Gebauer and Skerra, Engineered protein scaffolds as next-generationantibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumppet al., Darpins: A new generation of protein therapeutics. DrugDiscovery Today 13: 695-701 (2008). In one aspect of the invention, ascaffold antigen binding protein is selected from the group consistingof CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derivedmolecule such as Z-domain of Protein A (Affibody), an A-domain(Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrinrepeat protein (DARPin), a variable domain of antibody light chain orheavy chain (single-domain antibody, sdAb), a variable domain ofantibody heavy chain (nanobody, aVH), V_(NAR) fragments, a fibronectin(AdNectin), a C-type lectin domain (Tetranectin); a variable domain of anew antigen receptor beta-lactamase (V_(NAR) fragments), a humangamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domainof human protease inhibitors, microbodies such as the proteins from theknottin family, peptide aptamers and fibronectin (adnectin). CTLA-4(Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-family receptorexpressed on mainly CD4+ T-cells. Its extracellular domain has avariable domain-like Ig fold. Loops corresponding to CDRs of antibodiescan be substituted with heterologous sequence to confer differentbinding properties. CTLA-4 molecules engineered to have differentbinding specificities are also known as Evibodies (e.g. U.S. Pat. No.7,166,697B1). Evibodies are around the same size as the isolatedvariable region of an antibody (e.g. a domain antibody). For furtherdetails see Journal of Immunological Methods 248 (1-2), 31-45 (2001).Lipocalins are a family of extracellular proteins which transport smallhydrophobic molecules such as steroids, bilins, retinoids and lipids.They have a rigid beta-sheet secondary structure with a number of loopsat the open end of the conical structure which can be engineered to bindto different target antigens. Anticalins are between 160-180 amino acidsin size, and are derived from lipocalins. For further details seeBiochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 andUS20070224633. An affibody is a scaffold derived from Protein A ofStaphylococcus aureus which can be engineered to bind to antigen. Thedomain consists of a three-helical bundle of approximately 58 aminoacids. Libraries have been generated by randomization of surfaceresidues. For further details see Protein Eng. Des. Sel. 2004, 17,455-462 and EP 1641818A1. Avimers are multidomain proteins derived fromthe A-domain scaffold family. The native domains of approximately 35amino acids adopt a defined disulfide bonded structure. Diversity isgenerated by shuffling of the natural variation exhibited by the familyof A-domains. For further details see Nature Biotechnology 23(12),1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6),909-917 (June 2007). A transferrin is a monomeric serum transportglycoprotein. Transferrins can be engineered to bind different targetantigens by insertion of peptide sequences in a permissive surface loop.Examples of engineered transferrin scaffolds include the Trans-body. Forfurther details see J. Biol. Chem 274, 24066-24073 (1999). DesignedAnkyrin Repeat Proteins (DARPins) are derived from Ankyrin which is afamily of proteins that mediate attachment of integral membrane proteinsto the cytoskeleton. A single ankyrin repeat is a 33 residue motifconsisting of two alpha-helices and a beta-turn. They can be engineeredto bind different target antigens by randomizing residues in the firstalpha-helix and a beta-turn of each repeat. Their binding interface canbe increased by increasing the number of modules (a method of affinitymaturation). For further details see J. Mol. Biol. 332, 489-503 (2003),PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007)and US20040132028A1.

A single-domain antibody is an antibody fragment consisting of a singlemonomeric variable antibody domain. The first single domains werederived from the variable domain of the antibody heavy chain fromcamelids (nanobodies or V_(H)H fragments). Furthermore, the termsingle-domain antibody includes an autonomous human heavy chain variabledomain (aVH) or V_(NAR) fragments derived from sharks. Fibronectin is ascaffold which can be engineered to bind to antigen. Adnectins consistsof a backbone of the natural amino acid sequence of the 10th domain ofthe 15 repeating units of human fibronectin type III (FN3). Three loopsat one end of the .beta.-sandwich can be engineered to enable anAdnectin to specifically recognize a therapeutic target of interest. Forfurther details see Protein Eng. Des. Sel. 18, 435-444 (2005),US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1. Peptideaptamers are combinatorial recognition molecules that consist of aconstant scaffold protein, typically thioredoxin (TrxA) which contains aconstrained variable peptide loop inserted at the active site. Forfurther details see Expert Opin. Biol. Ther. 5, 783-797 (2005).Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges—examples ofmicroproteins include KalataBI and conotoxin and knottins. Themicroproteins have a loop which can beengineered to include upto 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see WO2008098796.

An “antigen binding molecule that binds to the same epitope” as areference molecule refers to an antigen binding molecule that blocksbinding of the reference molecule to its antigen in a competition assayby 50% or more, and conversely, the reference molecule blocks binding ofthe antigen binding molecule to its antigen in a competition assay by50% or more.

As used herein, the term “antigen binding domain” or “antigen-bindingsite” refers to the part of the antigen binding molecule thatspecifically binds to an antigenic determinant. More particularly, theterm “antigen-binding domain” refers the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. Where an antigen is large, an antigen bindingmolecule may only bind to a particular part of the antigen, which partis termed an epitope. An antigen binding domain may be provided by, forexample, one or more variable domains (also called variable regions).Preferably, an antigen binding domain comprises an antibody light chainvariable region (VL) and an antibody heavy chain variable region (VH).In one aspect, the antigen binding domain is able to bind to its antigenand block or partly block its function. Antigen binding domains thatspecifically bind to PD1 or to LAG3 include antibodies and fragmentsthereof as further defined herein. In addition, antigen binding domainsmay include scaffold antigen binding proteins, e.g. binding domainswhich are based on designed repeat proteins or designed repeat domains(see e.g. WO 2002/020565).

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins useful as antigens herein canbe any native form the proteins from any vertebrate source, includingmammals such as primates (e.g. humans) and rodents (e.g. mice and rats),unless otherwise indicated. In a particular embodiment the antigen is ahuman protein. Where reference is made to a specific protein herein, theterm encompasses the “full-length”, unprocessed protein as well as anyform of the protein that results from processing in the cell. The termalso encompasses naturally occurring variants of the protein, e.g.splice variants or allelic variants.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding molecule to bind to aspecific antigen can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed ona BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)),and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

In one embodiment, the extent of binding of an antigen binding moleculeto an unrelated protein is less than about 10% of the binding of theantigen binding molecule to the antigen as measured, e.g. by SPR. Incertain embodiments, an molecule that binds to the antigen has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁷ M or less, e.g. from 10⁻⁷ M to 10⁻¹³M, e.g. from 10⁻⁹ M to 10⁻¹³ M).

“Affinity” or “binding affinity” refers to the strength of the sum totalof non-covalent interactions between a single binding site of a molecule(e.g. an antibody) and its binding partner (e.g. an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g. antibody and antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (Kd), which is the ratio of dissociation andassociation rate constants (koff and kon, respectively). Thus,equivalent affinities may comprise different rate constants, as long asthe ratio of the rate constants remains the same. Affinity can bemeasured by common methods known in the art, including those describedherein. A particular method for measuring affinity is Surface PlasmonResonance (SPR).

As used herein, the term “high affinity” of an antibody refers to anantibody having a Kd of 10⁻⁹ M or less and even more particularly 10⁻¹⁰M or less for a target antigen. The term “low affinity” of an antibodyrefers to an antibody having a Kd of 10⁻⁸ or higher.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “a bispecific antibody comprising a first antigen bindingdomain that specifically binds to PD1 and a second antigen bindingdomain that specifically binds to LAG3” “a bispecific antibody thatspecifically binds PD1 and LAG3”, “bispecific antigen binding moleculespecific for PD1 and LAG3” or an “anti-PD1/anti-LAG3 antibody” are usedinterchangeably herein and refer to a bispecific antibody that iscapable of binding PD1 and LAG3 with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetingPD1 and LAG3.

The term “PD1”, also known as Programmed cell death protein 1, is a typeI membrane protein of 288 amino acids that was first described in 1992(Ishida et al., EMBO J., 11 (1992), 3887-3895). PD-1 is a member of theextended CD28/CTLA-4 family of T cell regulators and has two ligands,PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The protein's structureincludes an extracellular IgV domain followed by a transmembrane regionand an intracellular tail. The intracellular tail contains twophosphorylation sites located in an immunoreceptor tyrosine-basedinhibitory motif and an immunoreceptor tyrosine-based switch motif,which suggests that PD-1 negatively regulates TCR signals. This isconsistent with binding of SHP-1 and SHP-2 phosphatases to thecytoplasmic tail of PD-1 upon ligand binding. While PD-1 is notexpressed on naïve T cells, it is upregulated following T cell receptor(TCR)-mediated activation and is observed on both activated andexhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772).These exhausted T-cells have a dysfunctional phenotype and are unable torespond appropriately. Although PD-1 has a relatively wide expressionpattern its most important role is likely as a coinhibitory receptor onT cells (Chinai et al, Trends in Pharmacological Sciences 36 (2015),587-595). Current therapeutic approaches thus focus on blocking theinteraction of PD-1 with its ligands to enhance T cell response. Theterms “Programmed Death 1,” “Programmed Cell Death 1,” “Protein PD-1,”“PD-1,” PD1,” “PDCD1,” “hPD-1” and “hPD-1” can be used interchangeably,and include variants, isoforms, species homologs of human PD-1, andanalogs having at least one common epitope with PD-1. The amino acidsequence of human PD1 is shown in UniProt (www.uniprot.org) accessionno. Q15116 (SEQ ID NO: 128).

The terms “anti-PD1 antibody” and “an antibody comprising an antigenbinding domain that binds to PD1” refer to an antibody that is capableof binding PD1, especially a PD1 polypeptide expressed on a cellsurface, with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting PD1. In one aspect, theextent of binding of an anti-PD1 antibody to an unrelated, non-PD1protein is less than about 10% of the binding of the antibody to PD1 asmeasured, e.g., by radioimmunoassay (RIA) or flow cytometry (FACS) or bya Surface Plasmon Resonance assay using a biosensor system such as aBiacore) system. In certain aspects, an antigen binding protein thatbinds to human PD1 has a K_(D) value of the binding affinity for bindingto human PD1 of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M). In one preferred embodiment the respective K_(D)value of the binding affinities is determined in a Surface PlasmonResonance assay using the Extracellular domain (ECD) of human PD1(PD1-ECD) for the PD1 binding affinity. The term “anti-PD1 antibody”also encompasses bispecific antibodies that are capable of binding PD1and a second antigen.

The terms “LAG3” or “Lag-3” or “Lymphocyte activation gene-3” or “CD223”as used herein refer to any native LAG3 from any vertebrate source,including mammals such as primates (e.g. humans) and rodents (e.g., miceand rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed LAG3 as well as any form of LAG3 resultingfrom processing in the cell. The term also encompasses naturallyoccurring variants of LAG3, e.g., splice variants or allelic variants.In one preferred embodiment the term “LAG3” refers to human LAG3. Theamino acid sequence of an exemplary processed (without signal sequences)LAG3 is shown in SEQ ID NO:73. The amino acid sequence of an exemplaryExtracellular Domain (ECD) LAG3 is shown in SEQ ID NO:74.

The terms “anti-LAG3 antibody” and “an antibody that binds to LAG3”refer to an antibody that is capable of binding LAG3 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting LAG3. In one aspect, the extent ofbinding of an anti-LAG3 antibody to an unrelated, non-LAG3 protein isless than about 10% of the binding of the antibody to LAG3 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to LAG3 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less,e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainaspects, an anti-LAG3 antibody binds to an epitope of LAG3 that isconserved among LAG3 from different species. In one preferredembodiment, an “anti-LAG3 antibody”, “an antibody that specificallybinds to human LAG3”, and “an antibody that binds to human LAG3” refersto an antibody specifically binding to the human LAG3 antigen or itsExtracellular Domain (ECD) with a binding affinity of a K_(D)-value of1.0×10⁻⁸ mol/l or lower, in one embodiment of a K_(D)-value of 1.0×10⁻⁹mol/l or lower, in one embodiment of a K_(D)-value of 1.0×10⁻⁹ mol/l to1.0×10⁻¹³ mol/l. In this context the binding affinity is determined witha standard binding assay, such as surface plasmon resonance technique(BIAcore®, GE-Healthcare Uppsala, Sweden) e.g. using the LAG3extracellular domain. The term “anti-LAG3 antibody” also encompassesbispecific antibodies that are capable of binding LAG3 and a secondantigen.

A “blocking” antibody or an “antagonist” antibody is one that inhibitsor reduces a biological activity of the antigen it binds. In someembodiments, blocking antibodies or antagonist antibodies substantiallyor completely inhibit the biological activity of the antigen. Forexample, the bispecific antibodies of the invention block the signalingthrough PD-1 and LAG3 so as to restore a functional response by T cells(e.g., proliferation, cytokine production, target cell killing) from adysfunctional state to antigen stimulation.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding the antigenbinding molecule to antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three hypervariable regions (HVRs). See,e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page91 (2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR,” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1. L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) Hypervariable regions(HVRs) are also referred to as complementarity determining regions(CDRs), and these terms are used herein interchangeably in reference toportions of the variable region that form the antigen binding regions.This particular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of Proteins of ImmunologicalInterest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917(1987), where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table A as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35  26-32 26-35  V_(H) CDR2 50-65  52-58  50-58  V_(H) CDR3 95-102 95-102 95-102V_(L) CDR1 24-34  26-32  24-34  V_(L) CDR2 50-56  50-52  50-56  V_(L)CDR3 89-97  91-96  89-97  ¹Numbering of all CDR definitions in Table Ais according to the numbering conventions set forth by Kabat et al. (seebelow). ²″AbM″ with a lowercase “b” as used in Table A refers to theCDRs as defined by Oxford Molecular's ″AbM″ antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. CDRs also comprise“specificity determining residues,” or “SDRs,” which are residues thatcontact antigen. SDRs are contained within regions of the CDRs calledabbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2,a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633(2008).) Unless otherwise indicated. HVR residues and other residues inthe variable domain (e.g., FR residues) are numbered herein according toKabat et al.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g. IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ respectively.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody.

A humanized antibody optionally may comprise at least a portion of anantibody constant region derived from a human antibody. A “humanizedform” of an antibody, e.g., a non-human antibody, refers to an antibodythat has undergone humanization. Other forms of “humanized antibodies”encompassed by the present invention are those in which the constantregion has been additionally modified or changed from that of theoriginal antibody to generate the properties according to the invention,especially in regard to C1q binding and/or Fc receptor (FcR) binding.

A “human” antibody is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an antibody heavy chain that contains at least aportion of the constant region. The term includes native sequence Fcregions and variant Fc regions. Particularly, a human IgG heavy chain Fcregion extends from Cys226, or from Pro230, to the carboxyl-terminus ofthe heavy chain. However, the C-terminal lysine (Lys447) of the Fcregion may or may not be present. The amino acid sequences of the heavychains are always presented with the C-terminal lysine, however variantswithout the C-terminal lysine are included in the invention.

An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain. The “CH2domain” of a human IgG Fc region usually extends from an amino acidresidue at about position 231 to an amino acid residue at about position340. In one embodiment, a carbohydrate chain is attached to the CH2domain. The CH2 domain herein may be a native sequence CH2 domain orvariant CH2 domain. The “CH3 domain” comprises the stretch of residuesC-terminal to a CH2 domain in an Fc region (i.e. from an amino acidresidue at about position 341 to an amino acid residue at about position447 of an IgG). The CH3 region herein may be a native sequence CH3domain or a variant CH3 domain (e.g. a CH3 domain with an introduced“protuberance” (“knob”) in one chain thereof and a correspondingintroduced “cavity” (“hole”) in the other chain thereof; see U.S. Pat.No. 5,821,333, expressly incorporated herein by reference). Such variantCH3 domains may be used to promote heterodimerization of twonon-identical antibody heavy chains as herein described. Unlessotherwise specified herein, numbering of amino acid residues in the Fcregion or constant region is according to the EU numbering system, alsocalled the EU index, as described in Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991.

The “knob-into-hole” technology is described e.g. in U.S. Pat. No.5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, themethod involves introducing a protuberance (“knob”) at the interface ofa first polypeptide and a corresponding cavity (“hole”) in the interfaceof a second polypeptide, such that the protuberance can be positioned inthe cavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). The protuberance and cavitycan be made by altering the nucleic acid encoding the polypeptides, e.g.by site-specific mutagenesis, or by peptide synthesis. In a specificembodiment a knob modification comprises the amino acid substitutionT366W in one of the two subunits of the Fc domain, and the holemodification comprises the amino acid substitutions T366S, L368A andY407V in the other one of the two subunits of the Fc domain. In afurther specific embodiment, the subunit of the Fc domain comprising theknob modification additionally comprises the amino acid substitutionS354C, and the subunit of the Fc domain comprising the hole modificationadditionally comprises the amino acid substitution Y349C. Introductionof these two cysteine residues results in the formation of a disulfidebridge between the two subunits of the Fc region, thus furtherstabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

A “region equivalent to the Fc region of an immunoglobulin” is intendedto include naturally occurring allelic variants of the Fc region of animmunoglobulin as well as variants having alterations which producesubstitutions, additions, or deletions but which do not decreasesubstantially the ability of the immunoglobulin to mediate effectorfunctions (such as antibody-dependent cellular cytotoxicity). Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity(see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CDI6a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (seeUniProt accession no. P08637, version 141).

The term “peptide linker” refers to a peptide comprising one or moreamino acids, typically about 2 to 20 amino acids. Peptide linkers areknown in the art or are described herein. Suitable, non-immunogeniclinker peptides are, for example, (G₄S)_(n), (SG₄)_(n) or G₄(SG₄)_(n)peptide linkers, wherein “n” is generally a number between 1 and 10,typically between 2 and 4, in particular 2, i.e. the peptides selectedfrom the group consisting of GGGGS (SEQ ID NO: 129) GGGGSGGGGS (SEQ IDNO: 130), SGGGGSGGGG (SEQ ID NO: 131) and GGGGSGGGGSGGGG (SEQ ID NO:132), but also include the sequences GSPGSSSSGS (SEQ ID NO: 133), (G4S)₃(SEQ ID NO: 134), (G4S)₄ (SEQ ID NO: 135). GSGSGSGS (SEQ ID NO: 136),GSGSGNGS (SEQ ID NO: 137), GGSGSGSG (SEQ ID NO: 138), GGSGSG (SEQ ID NO:139), GGSG (SEQ ID NO: 140), GGSGNGSG (SEQ ID NO: 141), GGNGSGSG (SEQ IDNO:142) and GGNGSG (SEQ ID NO: 143). Peptide linkers of particularinterest are (G4S) (SEQ ID NO:129), (G₄S)₂ or GGGGSGGGGS (SEQ ID NO:130), (G4S)₃ (SEQ ID NO:134) and (G₄S)₄ (SEQ ID NO: 135), moreparticularly (G₄S)₂ or GGGGSGGGGS (SEQ ID NO: 130).

By “fused to” or “connected to” is meant that the components (e.g. anantigen binding domain and a FC domain) are linked by peptide bonds,either directly or via one or more peptide linkers.

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide (protein) sequence is defined as the percentage of aminoacid residues in a candidate sequence that are identical with the aminoacid residues in the reference polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN, SAWIor Megalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc., and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available from Genentech,Inc., South San Francisco, Calif. or may be compiled from the sourcecode. The ALIGN-2 program should be compiled for use on a UNIX operatingsystem, including digital UNIX V4.0D. All sequence comparison parametersare set by the ALIGN-2 program and do not vary. In situations whereALIGN-2 is employed for amino acid sequence comparisons, the % aminoacid sequence identity of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

In certain aspects, amino acid sequence variants of the bispecificantibodies of the invention provided herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the bispecific antibodies. Amino acidsequence variants of the bispecific antibodies may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the molecules, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.Sites of interest for substitutional mutagenesis include the HVRs andFramework (FRs). Conservative substitutions are provided in Table Bunder the heading “Preferred Substitutions” and further described belowin reference to amino acid side chain classes (1) to (6). Amino acidsubstitutions may be introduced into the molecule of interest and theproducts screened for a desired activity. e.g., retained/improvedantigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE B Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala. Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “amino acid sequence variants” includes substantial variantswherein there are amino acid substitutions in one or more hypervariableregion residues of a parent antigen binding molecule (e.g. a humanizedor human antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antigen binding molecule and/or will havesubstantially retained certain biological properties of the parentantigen binding molecule. An exemplary substitutional variant is anaffinity matured antibody, which may be conveniently generated, e.g.,using phage display-based affinity maturation techniques such as thosedescribed herein. Briefly, one or more HVR residues are mutated and thevariant antigen binding molecules displayed on phage and screened for aparticular biological activity (e.g. binding affinity). In certainembodiments, substitutions, insertions, or deletions may occur withinone or more HVRs so long as such alterations do not substantially reducethe ability of the antigen binding molecule to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. A useful method for identification of residues orregions of an antibody that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells(1989) Science, 244:1081-1085. In this method, a residue or group oftarget residues (e.g., charged residues such as Arg, Asp, His, Lys, andGlu) are identified and replaced by a neutral or negatively chargedamino acid (e.g., alanine or polyalanine) to determine whether theinteraction of the antibody with antigen is affected. Furthersubstitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antigen binding molecule complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues may be targeted or eliminated as candidates for substitution.Variants may be screened to determine whether they contain the desiredproperties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includebispecific antibodies with an N-terminal methionyl residue. Otherinsertional variants of the molecule include the fusion to the N- orC-terminus to a polypeptide which increases the serum half-life of thebispecific antibody.

In certain aspects, the bispecific antibodies provided herein arealtered to increase or decrease the extent to which the antibody isglycosylated. Glycosylation variants of the molecules may beconveniently obtained by altering the amino acid sequence such that oneor more glycosylation sites is created or removed, e.g. thecarbohydrates attached to the Fc domain may be altered. Nativeantibodies produced by mammalian cells typically comprise a branched,biantennary oligosaccharide that is generally attached by an N-linkageto Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.TIBTECH 15:26-32 (1997). The oligosaccharide may include variouscarbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose,and sialic acid, as well as a fucose attached to a GlcNAc in the “stem”of the biantennary oligosaccharide structure. In some embodiments,modifications of the oligosaccharide in the bispecific antibodies of theinvention may be made in order to create variants with certain improvedproperties. In one aspect, variants of bispecific antibodies areprovided having a carbohydrate structure that lacks fucose attached(directly or indirectly) to an Fc region. Such fucosylation variants mayhave improved ADCC function, see e.g. US Patent Publication Nos. US2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co.,Ltd). Further variants of the bispecific antibodies of the inventioninclude those with bisected oligosaccharides, e.g., in which abiantennary oligosaccharide attached to the Fc region is bisected byGlcNAc. Such variants may have reduced fucosylation and/or improved ADCCfunction, see for example WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).Variants with at least one galactose residue in the oligosaccharideattached to the Fc region are also provided. Such antibody variants mayhave improved CDC function and are described, e.g., in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju. S.); and WO 1999/22764 (Raju, S.).

In certain aspects, it may be desirable to create cysteine engineeredvariants of the bispecific antibodies of the invention, e.g.,“thioMAbs,” in which one or more residues of the molecule aresubstituted with cysteine residues. In particular embodiments, thesubstituted residues occur at accessible sites of the molecule. Bysubstituting those residues with cysteine, reactive thiol groups arethereby positioned at accessible sites of the antibody and may be usedto conjugate the antibody to other moieties, such as drug moieties orlinker-drug moieties, to create an immunoconjugate. In certainembodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of theheavy chain Fc region. Cysteine engineered antigen binding molecules maybe generated as described, e.g., in U.S. Pat. No. 7,521,541.

In certain aspects, the bispecific antibodies provided herein may befurther modified to contain additional non-proteinaceous moieties thatare known in the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether thebispecific antibody derivative will be used in a therapy under definedconditions, etc.

In another aspect, conjugates of an antibody and non-proteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the non-proteinaceous moiety is a carbonnanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells.” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe bispecific antigen binding molecules of the present invention. Inparticular, the host cell is a prokaryotic or eukaryotic host cell. Hostcells include cultured cells, e.g. mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable excipient” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable excipient includes,but is not limited to, a buffer, a stabilizer, or a preservative.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein. “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, the moleculesof the invention are used to delay development of a disease or to slowthe progression of a disease.

The term “cancer” as used herein refers to proliferative diseases, suchas lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliay cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma and Ewings sarcoma, including refractory versions ofany of the above cancers, or a combination of one or more of the abovecancers.

Bispecific Antibodies of the Invention

The invention provides novel bispecific antibodies comprising a firstantigen binding domain that specifically binds to programmed cell deathprotein 1 (PD1) and a second antigen binding domain that specificallybinds to Lymphocyte activation gene-3 (LAG3), with particularlyadvantageous properties such as producibility, stability, bindingaffinity, biological activity, specific targeting of certain T cells,targeting efficiency and reduced toxicity.

In certain aspects, a bispecific antibody comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 is provided that showsreduced internalization upon binding to the T cell surface. Theinternalization represents an important sink for the molecule which canbe degraded within a few hours while the targeted receptors are rapidlyre-expressed on the cell-surface ready to inhibit TCR-signalling. Infurther aspects, a bispecific antibody comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 is provided thatpreferentially binds to conventional T cells rather than to Tregs.

This is advantageous because targeting LAG-3 on Tregs with blockingantibodies could be detrimental by increasing their suppressive functionand eventually mask the positive blocking effect on other T cells. In afurther aspect, a bispecific antibody comprising a first antigen bindingdomain that specifically binds to PD1 and a second antigen bindingdomain that specifically binds to LAG3 is provided that is able torescue T cell effector functions from Treg suppression. In anotheraspect, a bispecific antibody comprising a first antigen binding domainthat specifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 is provided that is able to induce Granzyme Bsecretion by CD4 T cells, when co-cultured with the tumor cell lineARH77 as shown in the assay provided herein. In a further aspect, abispecific antibody comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 is provided that shows increasedtumor-specific T cell effector functions and/or enhances the cytotoxiceffect of T cells. In another aspect, a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 is provided thatshows increased tumor eradication in vivo.

A. Exemplary Bispecific Antibodies that Bind to PD1 and LAG3

In one aspect, the invention provides a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein saidfirst antigen binding domain specifically binding to PD1 comprises

-   a VH domain comprising-   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1,-   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and-   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:3; and-   a VL domain comprising-   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4;-   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and-   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:6.

In one aspect, the bispecific antibody comprises a Fc domain that is anIgG, particularly an IgG1 Fc domain or an IgG4 Fc domain and wherein theFc domain has reduced or even abolished effector function. Inparticular, the Fc domain comprises one or more amino acid substitutionthat reduces binding to an Fc receptor, in particular towards Fcγreceptor.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises a Fc domain that is an IgG, particularlyan IgG1 Fc domain or an IgG4 Fc domain and wherein the Fc domaincomprises one or more amino acid substitution that reduces binding to anFc receptor, in particular towards Fcγ receptor.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thesecond antigen binding domain that specifically binds to LAG3 comprises

-   -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:            14,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:            15, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:            16; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:            17,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:            18, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO: 19; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:22,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:23, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:24; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:25,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:26, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:27; or    -   (c) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:30,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:31, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:32; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:33,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:34, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:35; or    -   (d) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:38,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:39, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:40; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:41,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:42, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:43; or    -   (e) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:46,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:47, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:48; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:49,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:50, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:51.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thefirst antigen binding domain specifically binding to PD1 comprises

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        7 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 8, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 10, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 11, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 12, or    -   (e) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 13.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thefirst antigen binding domain that specifically binds to PD1 comprises

-   -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:80.        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:81, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:82; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:83,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:84, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:85; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:88,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:89, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:90; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:91,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:92, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:93.

In one aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thefirst antigen binding domain specifically binding to PD1 comprises

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        86 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 87, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        94 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 95.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thesecond antigen binding domain specifically binding to LAG3 comprises

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        20 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 21, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        28 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 29, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        36 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 37, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        44 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 45, or    -   (e) a VH domain comprising the amino acid sequence of SEQ ID NO:        52 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 53.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thesecond antigen binding domain that specifically binds to LAG3 comprisesa VH domain comprising

-   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:56,-   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:57, and-   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:58; and-   a VL domain comprising-   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:59,-   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:60, and-   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:61.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thesecond antigen binding domain specifically binding to LAG3 comprises

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        54 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 55, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        62 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 63, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        64 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 65, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        66 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 67.

In a particular aspect provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein

-   -   the first antigen binding domain specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 9 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 10,    -   and the second antigen binding domain specifically binding to        LAG3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 20 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 21 or a VH domain comprising the amino acid        sequence of SEQ ID NO: 52 and a VL domain comprising the amino        acid sequence of SEQ ID NO: 53.

In one aspect, the bispecific antibody of the invention comprises afirst antigen binding domain specifically binding to PD1 comprising a VHdomain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10 and a secondantigen binding domain specifically binding to LAG3 comprising a VHdomain comprising the amino acid sequence of SEQ ID NO: 20 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 21.

In a further aspect the bispecific antibody of the invention comprises afirst antigen binding domain specifically binding to PD1 comprising a VHdomain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10 and a secondantigen binding domain specifically binding to LAG3 comprising a VHdomain comprising the amino acid sequence of SEQ ID NO: 52 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 53.

In another aspect, the bispecific antibody of the invention comprises afirst antigen binding domain specifically binding to PD1 comprising a VHdomain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10 and a secondantigen binding domain specifically binding to LAG3 comprising a VHdomain comprising the amino acid sequence of SEQ ID NO: 62 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 63.

In yet another aspect, the bispecific antibody of the inventioncomprises a first antigen binding domain specifically binding to PD1comprising a VH domain comprising the amino acid sequence of SEQ ID NO:86 and a VL domain comprising the amino acid sequence of SEQ ID NO: 87and a second antigen binding domain specifically binding to LAG3comprising a VH domain comprising the amino acid sequence of SEQ ID NO:62 and a VL domain comprising the amino acid sequence of SEQ ID NO: 63.

In yet another aspect, the bispecific antibody of the inventioncomprises a first antigen binding domain specifically binding to PD1comprising a VH domain comprising the amino acid sequence of SEQ ID NO:94 and a VL domain comprising the amino acid sequence of SEQ ID NO: 95and a second antigen binding domain specifically binding to LAG3comprising a VH domain comprising the amino acid sequence of SEQ ID NO:62 and a VL domain comprising the amino acid sequence of SEQ ID NO: 63.

In a further aspect, the bispecific antibody comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 is a human, humanized orchimeric antibody. In particular, it is a humanized or chimericantibody.

In one aspect, the bispecific antibody comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 is bivalent. This meansthat the bispecific antibody comprises one antigen binding domain thatspecifically binds to PD1 and one antigen binding domain thatspecifically binds to LAG3 (1+1 format).

In one aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, a first Fab fragmentcomprising the antigen binding domain that specifically binds to PD1 anda second Fab fragment comprising the antigen binding domain thatspecifically binds to LAG3. In a particular aspect, in one of the Fabfragments the variable domains VL and VH are replaced by each other sothat the VH domain is part of the light chain and the VL domain is partof the heavy chain. In a particular aspect, in the first Fab fragmentcomprising the antigen binding domain that specifically binds to PD1 thevariable domains VL and VH are replaced by each other.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises

-   -   (a) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO: 96,        a first light chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 98,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            97, and a second light chain comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO:99, or    -   (b) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO: 96,        a first light chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 98,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            100, and a second light chain comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO: 101, or    -   (c) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        102, a first light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        104,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            103, and a second light chain comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO: 105, or    -   (d) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        106, a first light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        107,        -   a second heavy chain comprising an amino acid sequence with            at least 95% sequence identity to the sequence of SEQ ID NO:            103, and a second light chain comprising an amino acid            sequence with at least 95% sequence identity to the sequence            of SEQ ID NO: 105.

More particularly, the bispecific antibody comprises

-   -   (a) a first heavy chain comprising an amino acid sequence of SEQ        ID NO: 96, a first light chain comprising an amino acid sequence        of SEQ ID NO: 98,        -   a second heavy chain comprising an amino acid sequence of            SEQ ID NO: 97, and a second light chain comprising an amino            acid sequence of SEQ ID NO:99, or    -   (b) a first heavy chain comprising an amino acid sequence of SEQ        ID NO: 96, a first light chain comprising an amino acid sequence        of SEQ ID NO: 98,        -   a second heavy chain comprising an amino acid sequence of            SEQ ID NO: 100, and a second light chain comprising an amino            acid sequence of SEQ ID NO: 101, or    -   (c) a first heavy chain comprising an amino acid sequence of SEQ        ID NO: 102, a first light chain comprising an amino acid        sequence of SEQ ID NO: 104,        -   a second heavy chain comprising an amino acid sequence of            SEQ ID NO: 103, and a second light chain comprising an amino            acid sequence of SEQ ID NO: 105, or    -   (d) a first heavy chain comprising an amino acid sequence of SEQ        ID NO: 106, a first light chain comprising an amino acid        sequence of SEQ ID NO: 107,        -   a second heavy chain comprising an amino acid sequence of            SEQ ID NO: 103, and a second light chain comprising an amino            acid sequence of SEQ ID NO: 105.

More particularly, the bispecific antibody comprises a first heavy chaincomprising an amino acid sequence of SEQ ID NO: 96, a first light chaincomprising an amino acid sequence of SEQ ID NO: 98, a second heavy chaincomprising an amino acid sequence of SEQ ID NO: 100, and a second lightchain comprising an amino acid sequence of SEQ ID NO: 101.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, a first Fab fragmentcomprising the antigen binding domain that specifically binds to PD1 anda second Fab fragment comprising the antigen binding domain thatspecifically binds to LAG3 that is fused to the C-terminus of the Fcdomain. Particularly, the Fab fragment comprising the antigen bindingdomain that specifically binds to LAG3 is fused to the C-terminus of theFC domain via its VH domain (trans 1+1 format).

In a particular aspect, the bispecific antibody comprises a first heavychain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 96, a first light chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 98, a second heavy chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 144, and a second light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 101.More particularly, the bispecific antibody comprises a first heavy chaincomprising an amino acid sequence of SEQ ID NO: 96, a first light chaincomprising an amino acid sequence of SEQ ID NO: 98, a second heavy chaincomprising an amino acid sequence of SEQ ID NO: 144, and a second lightchain comprising an amino acid sequence of SEQ ID NO: 101.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, a first Fab fragmentcomprising the antigen binding domain that specifically binds to PD1, asecond Fab fragment comprising the antigen binding domain thatspecifically binds to LAG3 and a third Fab fragment comprising anantigen binding domain that specifically binds to LAG3. In a particularaspect, the the Fab fragment comprising the antigen binding domain thatspecifically binds to PD1 is fused via a peptide linker to theC-terminus of one of the heavy chains.

In this aspect, the bispecific antibody is trivalent with bivalentbinding to LAG3 and monovalent binding to PD1. This means that thebispecific antibody comprises one antigen binding domain thatspecifically binds to PD1 and two antigen binding domains thatspecifically bind to LAG3 (2+1 format).

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises

-   (a) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 118, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 115,    -   a second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 119,        and two second light chains comprising an amino acid sequence        with at least 95% sequence identity to the sequence of SEQ ID        NO: 101, or-   (b) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 120, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 115,    -   a second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 121,        and two second light chains comprising an amino acid sequence        with at least 95% sequence identity to the sequence of SEQ ID        NO:99, or-   (c) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 122, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 115,    -   a second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 103,        and two second light chains comprising an amino acid sequence        with at least 95% sequence identity to the sequence of SEQ ID        NO: 105.

More particularly, the bispecific antibody comprises

-   (a) a first heavy chain comprising an amino acid sequence of SEQ ID    NO: 118, a first light chain comprising an amino acid sequence of    SEQ ID NO: 115, a second heavy chain comprising an amino acid    sequence of SEQ ID NO: 119, and two second light chains comprising    an amino acid sequence of SEQ ID NO: 101, or-   (b) a first heavy chain comprising an amino acid sequence of SEQ ID    NO: 120, a first light chain comprising an amino acid sequence of    SEQ ID NO: 115, a second heavy chain comprising an amino acid    sequence of SEQ ID NO: 121, and two second light chains comprising    an amino acid sequence of SEQ ID NO:99, or-   (c) a first heavy chain comprising an amino acid sequence of SEQ ID    NO: 122, a first light chain comprising an amino acid sequence of    SEQ ID NO: 115, a second heavy chain comprising an amino acid    sequence of SEQ ID NO: 103, and two second light chains comprising    an amino acid sequence of SEQ ID NO: 105.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, a first Fab fragmentcomprising the antigen binding domain that specifically binds to PD1, asecond Fab fragment comprising the antigen binding domain thatspecifically binds to LAG3 and a third Fab fragment comprising anantigen binding domain that specifically binds to LAG3, wherein one ofthe Fab fragments comprising the antigen binding domain thatspecifically binds to LAG3 is fused via a peptide linker to theC-terminus of one of the heavy chains (trans 2+1 format).

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises a first heavy chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 96, a first light chain comprising an amino acid sequence with atleast 95% sequence identity to the sequence of SEQ ID NO: 98, a secondheavy chain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 145, and two second light chainscomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 101. More particularly, the bispecificantibody comprises a first heavy chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 96, afirst light chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 98, a second heavy chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 145, and two second light chains comprisingan amino acid sequence with at least 95% sequence identity to thesequence of SEQ ID NO: 101.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, two Fab fragments comprisingeach an antigen binding domain that specifically binds to LAG3 and asingle chain Fab (scFab) comprising the antigen binding domain thatspecifically binds to PD1. In particular, the scFab comprising anantigen binding domain that specifically binds to PD1 is fused via apeptide linker to the C-terminus to one of the heavy chains.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises

-   (a) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 123, a    second heavy chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 119, and two    light chains comprising each an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 101, or-   (b) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 124, a    second heavy chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 121, and two    light chains comprising each an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO:99, or-   (c) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 125, a    second heavy chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 103, and a    second light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO:105.

More particularly, the bispecific antibody comprises

-   (a) a first heavy chain comprising an amino acid sequence of SEQ ID    NO: 123, a second heavy chain comprising an amino acid sequence of    SEQ ID NO: 119, and two light chains comprising each an amino acid    sequence of SEQ ID NO: 101, or-   (b) a first heavy chain comprising an amino acid sequence of SEQ ID    NO: 124, a second heavy chain comprising an amino acid sequence of    SEQ ID NO: 121, and two light chains comprising each an amino acid    sequence of SEQ ID NO:99, or-   (c) a first heavy chain comprising an amino acid sequence of SEQ ID    NO: 125, a second heavy chain comprising an amino acid sequence of    SEQ ID NO: 103, and two light chains comprising each an amino acid    sequence of SEQ ID NO: 105.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, two Fab fragments comprisingeach an antigen binding domain that specifically binds to LAG3 and a VHand VL domain comprising the antigen binding domain that specificallybinds to PD1. In particular, the the VH domain of the antigen bindingdomain that specifically binds to PD1 is fused via a peptide linker tothe C-terminus of one of the heavy chains and the VL domain of theantigen binding domain that specifically binds to PD1 is fused via apeptide linker to the C-terminus of the other one of the heavy chains.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises a first heavy chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 126, a second heavy chain comprising an amino acid sequence withat least 95% sequence identity to the sequence of SEQ ID NO: 127, andtwo light chains comprising each an amino acid sequence with at least95% sequence identity to the sequence of SEQ ID NO: 109. Moreparticularly, the bispecific antibody comprises a first heavy chaincomprising an amino acid sequence of SEQ ID NO: 126, a second heavychain comprising an amino acid sequence of SEQ ID NO: 127, and two lightchains comprising each an amino acid sequence of SEQ ID NO: 109.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, a first Fab fragmentcomprising the antigen binding domain that specifically binds to PD1, asecond Fab fragment comprising the antigen binding domain thatspecifically binds to LAG3, a third Fab fragment comprising an antigenbinding domain that specifically binds to LAG3, and a fourth Fabfragment comprising an antigen binding domain that specifically binds toPD1.

In this aspect, the bispecific antibody is tetravalent with bivalentbinding to LAG3 and bivalent binding to PD1. This means that thebispecific antibody comprises two antigen binding domains thatspecifically bind to PD1 and two antigen binding domains thatspecifically bind to LAG3 (2+2 format).

In one aspect, the bispecific antibody of the invention comprises

-   (a) two light chains and two heavy chains of an antibody comprising    two Fab fragments comprising the antigen binding domains that    specifically bind to LAG3, and-   (b) two additional Fab fragments comprising the antigen binding    domains that specifically bind to PD1, wherein said additional Fab    fragments are each connected via a peptide linker to the C-terminus    of the heavy chains of (a).

In a particular aspect, the peptide linker is (G₄S)₄. In another aspect,the two additional Fab fragments comprising the antigen binding domainsthat specifically bind to PD1 are crossover Fab fragments wherein thevariable domains VL and VH are replaced by each other and the VL-CHchains are each connected via a peptide linker to the C-terminus of theheavy chains of (a).

In one aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein the twoFab fragments comprising each an antigen binding domain thatspecifically binds to PD1 are each fused via a peptide linker to theC-terminus to one of the heavy chains, respectively.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises

-   (a) two heavy chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 114, two    first light chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 115, and    two second light chains comprising each an amino acid sequence with    at least 95% sequence identity to the sequence of SEQ ID NO: 101, or-   (b) two heavy chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 116, two    first light chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 115, and    two second light chains comprising each an amino acid sequence with    at least 95% sequence identity to the sequence of SEQ ID NO:99, or-   (c) two heavy chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 117, two    first light chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 115, and    two second light chains comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 105.

More particularly, the bispecific antibody comprises

-   (a) two heavy chains comprising each an amino acid sequence of SEQ    ID NO: 114, two first light chains comprising each an amino acid    sequence of SEQ ID NO: 115, and two second light chains comprising    each an amino acid sequence of SEQ ID NO: 101, or-   (b) two heavy chains comprising each an amino acid sequence of SEQ    ID NO: 116, two first light chains comprising each an amino acid    sequence of SEQ ID NO: 115, and two second light chains comprising    each an amino acid sequence of SEQ ID NO:99, or-   (c) two heavy chains comprising each an amino acid sequence of SEQ    ID NO: 117, two first light chains comprising each an amino acid    sequence of SEQ ID NO: 115, and two second light chains comprising    an amino acid sequence of SEQ ID NO: 105.

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

In certain aspects, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises a Fc domain comprising one or more aminoacid modifications that reduce binding to an Fc receptor, in particulartowards Fcγ receptor, and reduce or abolish effector function.

In certain aspects, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

The following section describes preferred aspects of the bispecificantigen binding molecules of the invention comprising Fc domainmodifications reducing Fc receptor binding and/or effector function. Inone aspect, the invention relates to a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein the Fcdomain comprises one or more amino acid substitution that reducesbinding to an Fc receptor, in particular towards Fcγ receptor. Inparticular, the Fc domain is of human IgG1 subclass with the amino acidmutations L234A, L235A and P329G (numbering according to Kabat EUindex).

The Fc domain confers favorable pharmacokinetic properties to thebispecific antibodies of the invention, including a long serum half-lifewhich contributes to good accumulation in the target tissue and afavorable tissue-blood distribution ratio. At the same time it may,however, lead to undesirable targeting of the bispecific antibodies ofthe invention to cells expressing Fc receptors rather than to thepreferred antigen-bearing cells. Accordingly, in particular embodimentsthe Fc domain of the bispecific antibodies of the invention exhibitsreduced binding affinity to an Fc receptor and/or reduced effectorfunction, as compared to a native IgG Fc domain, in particular an IgG1Fc domain or an IgG4 Fc domain. More particularly, the Fc domain is anIgG1 FC domain.

In one such aspect the Fc domain (or the bispecific antigen bindingmolecule of the invention comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG1 Fc domain (or the bispecific antigen bindingmolecule of the invention comprising a native IgG1 Fc domain), and/orless than 50%, preferably less than 20%, more preferably less than 10%and most preferably less than 5% of the effector function, as comparedto a native IgG1 Fc domain (or the bispecific antigen binding moleculeof the invention comprising a native IgG1 Fc domain). In one aspect, theFc domain (or the bispecific antigen binding molecule of the inventioncomprising said Fc domain) does not substantially bind to an Fc receptorand/or induce effector function. In a particular aspect the Fc receptoris an Fcγ receptor. In one aspect, the Fc receptor is a human Fcreceptor. In one aspect, the Fc receptor is an activating Fc receptor.In a specific aspect, the Fc receptor is an activating human Fcγreceptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, mostspecifically human FcγRIIIa. In one aspect, the Fc receptor is aninhibitory Fc receptor. In a specific aspect, the Fc receptor is aninhibitory human Fcγ receptor, more specifically human FcγRIIB. In oneaspect the effector function is one or more of CDC, ADCC, ADCP, andcytokine secretion. In a particular aspect, the effector function isADCC. In one aspect, the Fc domain exhibits substantially similarbinding affinity to neonatal Fc receptor (FcRn), as compared to a nativeIgG1 Fc domain. Substantially similar binding to FcRn is achieved whenthe Fc domain (or the the bispecific antigen binding molecule of theinvention comprising said Fc domain) exhibits greater than about 70%,particularly greater than about 80%, more particularly greater thanabout 90% of the binding affinity of a native IgG1 Fc domain (or thebispecific antigen binding molecule of the invention comprising a nativeIgG1 Fc domain) to FcRn.

In a particular aspect, the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In a particular aspect, the Fcdomain of the bispecific antigen binding molecule of the inventioncomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function.Typically, the same one or more amino acid mutation is present in eachof the two subunits of the Fc domain. In one aspect, the amino acidmutation reduces the binding affinity of the Fc domain to an Fcreceptor. In another aspect, the amino acid mutation reduces the bindingaffinity of the Fc domain to an Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In one aspect, the bispecific antigenbinding molecule of the invention comprising an engineered Fc domainexhibits less than 20%, particularly less than 10%, more particularlyless than 5% of the binding affinity to an Fc receptor as compared tobispecific antibodies of the invention comprising a non-engineered Fcdomain. In a particular aspect, the Fc receptor is an Fcγ receptor. Inother aspects, the Fc receptor is a human Fc receptor. In one aspect,the Fc receptor is an inhibitory Fc receptor. In a specific aspect, theFc receptor is an inhibitory human Fcγ receptor, more specifically humanFcγRIIB. In some aspects the Fc receptor is an activating Fc receptor.In a specific aspect, the Fc receptor is an activating human Fcγreceptor, more specifically human FcγRIIIa. FcγRI or FcγRIIa, mostspecifically human FcγRIIIa Preferably, binding to each of thesereceptors is reduced. In some aspects, binding affinity to a complementcomponent, specifically binding affinity to C1q, is also reduced. In oneaspect, binding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn i.e. preservation of the bindingaffinity of the Fc domain to said receptor, is achieved when the Fcdomain (or the bispecific antigen binding molecule of the inventioncomprising said Fc domain) exhibits greater than about 70% of thebinding affinity of a non-engineered form of the Fc domain (or thebispecific antigen binding molecule of the invention comprising saidnon-engineered form of the Fc domain) to FcRn. The Fc domain, or thebispecific antigen binding molecule of the invention comprising said Fcdomain, may exhibit greater than about 80% and even greater than about90% of such affinity. In certain embodiments the Fc domain of thebispecific antigen binding molecule of the invention is engineered tohave reduced effector function, as compared to a non-engineered Fcdomain. The reduced effector function can include, but is not limitedto, one or more of the following: reduced complement dependentcytotoxicity (CDC), reduced antibody-dependent cell-mediatedcytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis(ADCP), reduced cytokine secretion, reduced immune complex-mediatedantigen uptake by antigen-presenting cells, reduced binding to NK cells,reduced binding to macrophages, reduced binding to monocytes, reducedbinding to polymorphonuclear cells, reduced direct signaling inducingapoptosis, reduced dendritic cell maturation, or reduced T cell priming.

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581). Certain antibody variants with improved or diminishedbinding to FcRs are described. (e.g. U.S. Pat. No. 6,737,056; WO2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276 (2001)6591-6604).

In one aspect of the invention, the Fc domain comprises an amino acidsubstitution at a position of E233, L234, L235, N297, P331 and P329. Insome aspects, the Fc domain comprises the amino acid substitutions L234Aand L235A (“LALA”). In one such embodiment, the Fc domain is an IgG1 Fcdomain, particularly a human IgG1 Fc domain. In one aspect, the Fcdomain comprises an amino acid substitution at position P329. In a morespecific aspect, the amino acid substitution is P329A or P329G,particularly P329G. In one embodiment the Fc domain comprises an aminoacid substitution at position P329 and a further amino acid substitutionselected from the group consisting of E233P, L234A, L235A, L235E, N297A,N297D or P331S. In more particular embodiments the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”). The“P329G LALA” combination of amino acid substitutions almost completelyabolishes Fcγ receptor binding of a human IgG1 Fc domain, as describedin PCT Patent Application No. WO 2012/130831 A1. Said document alsodescribes methods of preparing such mutant Fc domains and methods fordetermining its properties such as Fc receptor binding or effectorfunctions such antibody is an IgG1 with mutations L234A and L235A orwith mutations L234A, L235A and P329G (numbering according to EU indexof Kabat et al, Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991).

In one aspect, the bispecific antibody of the invention comprises (allpositions according to EU index of Kabat) (i) a homodimeric Fc-region ofthe human IgG1 subclass optionally with the mutations P329G, L234A andL235A, or (ii) a homodimeric Fc-region of the human IgG4 subclassoptionally with the mutations P329G, S228P and L235E, or (iii) ahomodimeric Fc-region of the human IgG1 subclass optionally with themutations P329G, L234A, L235A, I253A, H310A, and H435A, or optionallywith the mutations P329G, L234A, L235A, H310A, H433A, and Y436A, or (iv)a heterodimeric Fc-region wherein one Fc-region polypeptide comprisesthe mutation T366W, and the other Fc-region polypeptide comprises themutations T366S, L368A and Y407V, or wherein one Fc-region polypeptidecomprises the mutations T366W and Y349C, and the other Fc-regionpolypeptide comprises the mutations T366S, L368A, Y407V, and S354C, orwherein one Fc-region polypeptide comprises the mutations T366W andS354C, and the other Fc-region polypeptide comprises the mutationsT366S, L368A, Y407V and Y349C, or (v) a heterodimeric Fc-region of thehuman IgG1 subclass wherein both Fc-region polypeptides comprise themutations P329G, L234A and L235A and one Fc-region polypeptide comprisesthe mutation T366W, and the other Fc-region polypeptide comprises themutations T366S. L368A and Y407V, or wherein one Fc-region polypeptidecomprises the mutations T366W and Y349C, and the other Fc-regionpolypeptide comprises the mutations T366S, L368A, Y407V, and S354C, orwherein one Fc-region polypeptide comprises the mutations T366W andS354C, and the other Fc-region polypeptide comprises the mutationsT366S, L368A, Y407V and Y349C.

In one aspect, the Fc domain is an IgG4 Fc domain. In a more specificembodiment, the Fc domain is an IgG4 Fc domain comprising an amino acidsubstitution at position S228 (Kabat numbering), particularly the aminoacid substitution S228P. In a more specific embodiment, the Fc domain isan IgG4 Fc domain comprising amino acid substitutions L235E and S228Pand P329G. This amino acid substitution reduces in vivo Fab arm exchangeof IgG4 antibodies (see Stubenrauch et al., Drug Metabolism andDisposition 38, 84-91 (2010)). Thus, in one aspect, provided is abispecific antibody, comprising (all positions according to EU index ofKabat) a heterodimeric Fc-region of the human IgG4 subclass wherein bothFc-region polypeptides comprise the mutations P329G, S228P and L235E andone Fc-region polypeptide comprises the mutation T366W, and the otherFc-region polypeptide comprises the mutations T366S, L368A and Y407V, orwherein one Fc-region polypeptide comprises the mutations T366W andY349C, and the other Fc-region polypeptide comprises the mutationsT366S, L368A, Y407V, and S354C, or wherein one Fc-region polypeptidecomprises the mutations T366W and S354C, and the other Fc-regionpolypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan, A. R. and Winter. G., Nature 322 (1988) 738-740; U.S.Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerningother examples of Fc region variants.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor. Effector function of an Fc domain, or bispecific antibodies ofthe invention comprising an Fc domain, can be measured by methods knownin the art. A suitable assay for measuring ADCC is described herein.Other examples of in vitro assays to assess ADCC activity of a moleculeof interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al.Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., ProcNatl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337;Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.); and CytoTox 96® non-radioactivecytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g. in a animalmodel such as that disclosed in Clynes et al., Proc Natl Acad Sci USA95, 652-656 (1998).

The following section describes preferred aspects of the bispecificantibodies of the invention comprising Fc domain modifications reducingFc receptor binding and/or effector function. In one aspect, theinvention relates to the bispecific comprising a first antigen bindingdomain that specifically binds PD1 and a second antigen binding domainthat specifically binds to LAG3, wherein the Fc domain comprises one ormore amino acid substitution that reduces the binding affinity of theantibody to an Fc receptor, in particular towards Fcγ receptor. Inanother aspect, the invention relates to the bispecific antibodycomprising a first antigen binding domain that specifically binds to PD1and a second antigen binding domain that specifically binds to LAG3,wherein the Fc domain comprises one or more amino acid substitution thatreduces effector function. In particular aspect, the Fc domain is ofhuman IgG1 subclass with the amino acid mutations L234A, L235A and P329G(numbering according to Kabat EU index).

Fc Domain Modifications Promoting Heterodimerization

The bispecific antigen binding molecules of the invention comprisedifferent antigen binding domains, fused to one or the other of the twosubunits of the Fc domain, thus the two subunits of the Fc domain may becomprised in two non-identical polypeptide chains. Recombinantco-expression of these polypeptides and subsequent dimerization leads toseveral possible combinations of the two polypeptides. To improve theyield and purity of the bispecific antibodies of the invention inrecombinant production, it will thus be advantageous to introduce in theFc domain of the bispecific antigen binding molecules of the invention amodification promoting the association of the desired polypeptides.

Accordingly, in particular aspects the invention relates to a bispecificantibody comprising a first antigen binding domain that specificallybinds to PD1 and a second antigen binding domain that specifically bindsto LAG3, wherein the Fc domain comprises a modification promoting theassociation of the first and second subunit of the Fc domain. The siteof most extensive protein-protein interaction between the two subunitsof a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, inone aspect said modification is in the CH3 domain of the Fc domain.

In a specific aspect said modification is a so-called “knob-into-hole”modification, comprising a “knob” modification in one of the twosubunits of the Fc domain and a “hole” modification in the other one ofthe two subunits of the Fc domain. Thus, the invention relates to abispecific antibody comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to LAG3, wherein the first subunit of the Fc domaincomprises knobs and the second subunit of the Fc domain comprises holesaccording to the knobs into holes method. In a particular aspect, thefirst subunit of the Fc domain comprises the amino acid substitutionsS354C and T366W (EU numbering) and the second subunit of the Fc domaincomprises the amino acid substitutions Y349C, T366S and Y407V (numberingaccording to Kabat EU index).

The knob-into-hole technology is described e.g. in U.S. Pat. No.5,731,168; U.S. Pat. No. 7,695,936. Ridgway et al., Prot Eng 9, 617-621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, themethod involves introducing a protuberance (“knob”) at the interface ofa first polypeptide and a corresponding cavity (“hole”) in the interfaceof a second polypeptide, such that the protuberance can be positioned inthe cavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in one aspect, in the CH3 domain of the first subunit ofthe Fc domain of the bispecific antigen binding molecules of theinvention an amino acid residue is replaced with an amino acid residuehaving a larger side chain volume, thereby generating a protuberancewithin the CH3 domain of the first subunit which is positionable in acavity within the CH3 domain of the second subunit, and in the CH3domain of the second subunit of the Fc domain an amino acid residue isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity within the CH3 domain of the second subunitwithin which the protuberance within the CH3 domain of the first subunitis positionable. The protuberance and cavity can be made by altering thenucleic acid encoding the polypeptides, e.g. by site-specificmutagenesis, or by peptide synthesis. In a specific aspect, in the CH3domain of the first subunit of the Fc domain the threonine residue atposition 366 is replaced with a tryptophan residue (T366W), and in theCH3 domain of the second subunit of the Fc domain the tyrosine residueat position 407 is replaced with a valine residue (Y407V). In oneaspect, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A).

In yet a further aspect, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesleads to the formation of a disulfide bridge between the two subunits ofthe Fc domain, further stabilizing the dimer (Carter (2001), J ImmunolMethods 248, 7-15). In a particular aspect, the first subunit of the Fcdomain comprises the amino acid substitutions S354C and T366W (EUnumbering) and the second subunit of the Fc domain comprises the aminoacid substitutions Y349C, T366S and Y407V (numbering according to KabatEU index).

But also other knobs-in-holes technologies as described by EP 1 870 459,can be used alternatively or additionally. In one embodiment themultispecific antibody comprises the mutations R409D and K370E in theCH3 domain of the “knobs chain” and the mutations D399K and E357K in theCH3 domain of the “hole-chain” (numbering according to Kabat EU index).

In one aspect, the bispecific antibody comprises a T366W mutation in theCH3 domain of the “knobs chain” and the mutations T366S, L368A and Y407Vin the CH3 domain of the “hole chain” and additionally the mutationsR409D and K370E in the CH3 domain of the “knobs chain” and the mutationsD399K and E357K in the CH3 domain of the “hole chain” (numberingaccording to the Kabat EU index).

In one aspect, the bispecific antibody comprises the mutations Y349C andT366W in one of the two CH3 domains and the mutations S354C, T366S,L368A and Y407V in the other of the two CH3 domains, or themultispecific antibody comprises the mutations Y349C and T366W in one ofthe two CH3 domains and the mutations S354C, T366S. L368A and Y407V inthe other of the two CH3 domains and additionally the mutations R409Dand K370E in the CH3 domain of the “knobs chain” and the mutations D399Kand E357K in the CH3 domain of the “hole chain” (numbering according tothe Kabat EU index).

In an alternative aspect, a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

Apart from the “knob-into-hole technology” other techniques formodifying the CH3 domains of the heavy chains of a multispecificantibody to enforce heterodimerization are known in the art. Thesetechnologies, especially the ones described in WO 96/27011, WO98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004,WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO2013/157954 and WO 2013/096291 are contemplated herein as alternativesto the “knob-into-hole technology” in combination with a bispecificantibody.

In one aspect, in the bispecific antibody the approach described in EP1870459 is used to support heterodimerization of the first heavy chainand the second heavy chain of the multispecific antibody. This approachis based on the introduction of charged amino acids with oppositecharges at specific amino acid positions in the CH3/CH3-domain-interfacebetween both, the first and the second heavy chain.

Accordingly, in this aspect in the tertiary structure of themultispecific antibody the CH3 domain of the first heavy chain and theCH3 domain of the second heavy chain form an interface that is locatedbetween the respective antibody CH3 domains, wherein the respectiveamino acid sequences of the CH3 domain of the first heavy chain and theamino acid sequence of the CH3 domain of the second heavy chain eachcomprise a set of amino acids that is located within said interface inthe tertiary structure of the antibody, wherein from the set of aminoacids that is located in the interface in the CH3 domain of one heavychain a first amino acid is substituted by a positively charged aminoacid and from the set of amino acids that is located in the interface inthe CH3 domain of the other heavy chain a second amino acid issubstituted by a negatively charged amino acid. The bispecific antibodyaccording to this aspect is herein also referred to as“CH3(+/−)-engineered bispecific antibody” (wherein the abbreviation“+/−” stands for the oppositely charged amino acids that were introducedin the respective CH3 domains).

In one aspect, in the CH3(+/−)-engineered bispecific antibody thepositively charged amino acid is selected from K, R and H, and thenegatively charged amino acid is selected from E or D.

In one aspect, in the CH3(+/−)-engineered bispecific antibody thepositively charged amino acid is selected from K and R, and thenegatively charged amino acid is selected from E or D.

In one aspect, in the CH3(+/−)-engineered bispecific antibody thepositively charged amino acid is K, and the negatively charged aminoacid is E.

In one aspect, in the CH3(+/−)-engineered bispecific antibody in the CH3domain of one heavy chain the amino acid R at position 409 issubstituted by D and the amino acid K at position is substituted by E,and in the CH3 domain of the other heavy chain the amino acid D atposition 399 is substituted by K and the amino acid E at position 357 issubstituted by K (numbering according to Kabat EU index).

In one aspect, the approach described in WO 2013/157953 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one embodiment in the CH3 domainof one heavy chain the amino acid T at position 366 is substituted by K,and in the CH3 domain of the other heavy chain the amino acid L atposition 351 is substituted by D (numbering according to Kabat EUindex). In another embodiment in the CH3 domain of one heavy chain theamino acid T at position 366 is substituted by K and the amino acid L atposition 351 is substituted by K, and in the CH3 domain of the otherheavy chain the amino acid L at position 351 is substituted by D(numbering according to Kabat EU index).

In another aspect, in the CH3 domain of one heavy chain the amino acid Tat position 366 is substituted by K and the amino acid L at position 351is substituted by K, and in the CH3 domain of the other heavy chain theamino acid L at position 351 is substituted by D (numbering according toKabat EU index). Additionally at least one of the followingsubstitutions is comprised in the CH3 domain of the other heavy chain:the amino acid Y at position 349 is substituted by E, the amino acid Yat position 349 is substituted by D and the amino acid L at position 368is substituted by E (numbering according to Kabat EU index). In oneembodiment the amino acid L at position 368 is substituted by E(numbering according to Kabat EU index).

In one aspect, the approach described in WO 2012/058768 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one aspect, in the CH3 domain ofone heavy chain the amino acid L at position 351 is substituted by Y andthe amino acid Y at position 407 is substituted by A. and in the CH3domain of the other heavy chain the amino acid T at position 366 issubstituted by A and the amino acid K at position 409 is substituted byF (numbering according to Kabat EU index). In another embodiment, inaddition to the aforementioned substitutions, in the CH3 domain of theother heavy chain at least one of the amino acids at positions 411(originally T), 399 (originally D), 400 (originally S), 405 (originallyF), 390 (originally N) and 392 (originally K) is substituted (numberingaccording to Kabat EU index). Preferred substitutions are:

-   -   substituting the amino acid T at position 411 by an amino acid        selected from N, R, Q, K, D, E and W (numbering according to        Kabat EU index),    -   substituting the amino acid D at position 399 by an amino acid        selected from R, W, Y. and K (numbering according to Kabat EU        index),    -   substituting the amino acid S at position 400 by an amino acid        selected from E, D, R and K (numbering according to Kabat EU        index).    -   substituting the amino acid F at position 405 by an amino acid        selected from I, M, T, S, V and W (numbering according to Kabat        EU index;    -   substituting the amino acid N at position 390 by an amino acid        selected from R, K and D (numbering according to Kabat EU index;        and    -   substituting the amino acid K at position 392 by an amino acid        selected from V, M, R, L, F and E (numbering according to Kabat        EU index).

In another aspect, the bispecific antibody is engineered according to WO2012/058768), i.e. in the CH3 domain of one heavy chain the amino acid Lat position 351 is substituted by Y and the amino acid Y at position 407is substituted by A, and in the CH3 domain of the other heavy chain theamino acid T at position 366 is substituted by V and the amino acid K atposition 409 is substituted by F (numbering according to Kabat EUindex). In another embodiment of the multispecific antibody, in the CH3domain of one heavy chain the amino acid Y at position 407 issubstituted by A. and in the CH3 domain of the other heavy chain theamino acid T at position 366 is substituted by A and the amino acid K atposition 409 is substituted by F (numbering according to Kabat EUindex). In the last aforementioned embodiment, in the CH3 domain of theother heavy chain the amino acid K at position 392 is substituted by E,the amino acid T at position 411 is substituted by E, the amino acid Dat position 399 is substituted by R and the amino acid S at position 400is substituted by R (numbering according to Kabat EU index).

In one aspect, the approach described in WO 2011/143545 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one aspect, amino acidmodifications in the CH3 domains of both heavy chains are introduced atpositions 368 and/or 409 (numbering according to Kabat EU index).

In one aspect, the approach described in WO 2011/090762 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the bispecific antibody. WO 2011/090762 relates to amino acidmodifications according to the “knob-into-hole” (KiH) technology. In oneembodiment in the CH3 domain of one heavy chain the amino acid T atposition 366 is substituted by W, and in the CH3 domain of the otherheavy chain the amino acid Y at position 407 is substituted by A(numbering according to Kabat EU index). In another embodiment in theCH3 domain of one heavy chain the amino acid T at position 366 issubstituted by Y, and in the CH3 domain of the other heavy chain theamino acid Y at position 407 is substituted by T (numbering according toKabat EU index).

In one aspect, the approach described in WO 2009/089004 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the bispecific antibody. In one embodiment in the CH3 domain ofone heavy chain the amino acid K or N at position 392 is substituted bya negatively charged amino acid (in one embodiment by E or D, in onepreferred embodiment by D), and in the CH3 domain of the other heavychain the amino acid D at position 399 the amino acid E or D at position356 or the amino acid E at position 357 is substituted by a positivelycharged amino acid (in one embodiment K or R, in one preferredembodiment by K, in one preferred embodiment the amino acids atpositions 399 or 356 are substituted by K) (numbering according to KabatEU index). In one further embodiment, in addition to the aforementionedsubstitutions, in the CH3 domain of the one heavy chain the amino acid Kor R at position 409 is substituted by a negatively charged amino acid(in one embodiment by E or D, in one preferred embodiment by D)(numbering according to Kabat EU index). In one even further aspect inaddition to or alternatively to the aforementioned substitutions, in theCH3 domain of the one heavy chain the amino acid K at position 439and/or the amino acid K at position 370 is substituted independentlyfrom each other by a negatively charged amino acid (in one embodiment byE or D, in one preferred embodiment by D) (numbering according to KabatEU index).

In one aspect, the approach described in WO 2007/147901 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one embodiment in the CH3 domainof one heavy chain the amino acid K at position 253 is substituted by E,the amino acid D at position 282 is substituted by K and the amino acidK at position 322 is substituted by D, and in the CH3 domain of theother heavy chain the amino acid D at position 239 is substituted by K,the amino acid E at position 240 is substituted by K and the amino acidK at position 292 is substituted by D (numbering according to Kabat EUindex).

The C-terminus of the heavy chain of the bispecific antibody as reportedherein can be a complete C-terminus ending with the amino acid residuesPGK. The C-terminus of the heavy chain can be a shortened C-terminus inwhich one or two of the C terminal amino acid residues have beenremoved. In one preferred aspect, the C-terminus of the heavy chain is ashortened C-terminus ending PG.

In one aspect of all aspects as reported herein, a bispecific antibodycomprising a heavy chain including a C-terminal CH3 domain as specifiedherein, comprises the C-terminal glycine-lysine dipeptide (G446 andK447, numbering according to Kabat EU index). In one embodiment of allaspects as reported herein, a bispecific antibody comprising a heavychain including a C-terminal CH3 domain, as specified herein, comprisesa C-terminal glycine residue (G446, numbering according to Kabat EUindex).

Modifications in the Fab Domains

In one aspect, the invention relates to a bispecific antibody comprisinga first Fab fragment that specifically binds to PD1 and a second Fabfragment that specifically binds to LAG3, wherein in one of the Fabfragments either the variable domains VH and VL or the constant domainsCH1 and CL are exchanged. The bispecific antibodies are preparedaccording to the Crossmab technology.

Multispecific antibodies with a domain replacement/exchange in onebinding arm (CrossMabVH-VL or CrossMabCH-CL) are described in detail inWO2009/080252, WO2009/080253 and Schaefer, W. et al, PNAS, 108 (2011)11187-1191. They clearly reduce the byproducts caused by the mismatch ofa light chain against a first antigen with the wrong heavy chain againstthe second antigen (compared to approaches without such domainexchange).

In a particular aspect the invention relates to a bispecific antibodycomprising a first Fab fragment that specifically binds to PD1 and asecond Fab fragment that specifically binds to LAG3, wherein in one ofthe Fab fragments the variable domains VL and VH are replaced by eachother so that the VH domain is part of the light chain and the VL domainis part of the heavy chain. In a particular aspect, the bispecificantibody is one, wherein in the first Fab fragment comprising theantigen binding domain that specifically binds to PD1 the variabledomains VL and VH are replaced by each other.

In another aspect, and to further improve correct pairing, thebispecific antibody comprising a first Fab fragment that specificallybinds to PD1 and a second Fab fragment that specifically binds to LAG3,can contain different charged amino acid substitutions (so-called“charged residues”). These modifications are introduced in the crossedor non-crossed CH1 and CL domains. Such modifications are described e.g.in WO2015/150447, WO2016/020309 and PCT/EP2016/073408.

In a particular aspect, the invention is concerned with a bispecificantibody comprising a first Fab fragment that specifically binds to PD1and a second Fab fragment that specifically binds to LAG3, wherein inone of the Fab fragments in the constant domain CL the amino acid atposition 124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat EU Index), and in theconstant domain CH1 the amino acids at positions 147 and 213 aresubstituted independently by glutamic acid (E) or aspartic acid (D)(numbering according to Kabat EU index). In a particular aspect, thebispecific antibody is one, wherein in the second Fab fragmentcomprising the antigen binding domain that specifically binds to TIM3the constant domain CL the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat EU Index), and in the constant domain CH1 the aminoacids at positions 147 and 213 are substituted independently by glutamicacid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In a particular aspect, the invention relates to a bispecific antibodycomprising a first Fab fragment that specifically binds to PD1 and asecond Fab fragment that specifically binds to LAG3, wherein in one ofCL domains the amino acid at position 123 (EU numbering) has beenreplaced by arginine (R) and the amino acid at position 124 (EUnumbering) has been substituted by lysine (K) and wherein in one of theCH1 domains the amino acids at position 147 (EU numbering) and atposition 213 (EU numbering) have been substituted by glutamic acid (E).In a particular aspect, the bispecific antibody is one, wherein in thesecond Fab fragment comprising the antigen binding domain thatspecifically binds to LAG3 the amino acid at position 123 (EU numbering)has been replaced by arginine (R) and the amino acid at position 124 (EUnumbering) has been substituted by lysine (K) and wherein in one of theCH1 domains the amino acids at position 147 (EU numbering) and atposition 213 (EU numbering) have been substituted by glutamic acid (E).

In a further aspect, the bispecific antibody is a bivalent antibodycomprising

-   -   a) a first light chain and a first heavy chain of an antibody        specifically binding to a first antigen, and    -   b) a second light chain and a second heavy chain of an antibody        specifically binding to a second antigen, wherein the variable        domains VL and VH of the second light chain and the second heavy        chain are replaced by each other.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain under a) are isolated chains.

In the antibody under b) within the light chain the variable light chaindomain VL is replaced by the variable heavy chain domain VH of saidantibody, and within the heavy chain the variable heavy chain domain VHis replaced by the variable light chain domain VL of said antibody.

In one aspect, (i) in the constant domain CL of the first light chainunder a) the amino acid at position 124 (numbering according to Kabat)is substituted by a positively charged amino acid, and wherein in theconstant domain CH1 of the first heavy chain under a) the amino acid atposition 147 or the amino acid at position 213 (numbering according toKabat EU index) is substituted by a negatively charged amino acid, or(ii) in the constant domain CL of the second light chain under b) theamino acid at position 124 (numbering according to Kabat) is substitutedby a positively charged amino acid, and wherein in the constant domainCH1 of the second heavy chain under b) the amino acid at position 147 orthe amino acid at position 213 (numbering according to Kabat EU index)is substituted by a negatively charged amino acid.

In another aspect, (i) in the constant domain CL of the first lightchain under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and wherein in the constant domain CH1 of thefirst heavy chain under a) the amino acid at position 147 or the aminoacid at position 213 is substituted independently by glutamic acid (E)or aspartic acid (D) (numbering according to Kabat EU index), or (ii) inthe constant domain CL of the second light chain under b) the amino acidat position 124 is substituted independently by lysine (K), arginine (R)or histidine (H) (numbering according to Kabat) (in one preferredembodiment independently by lysine (K) or arginine (R)), and wherein inthe constant domain CH1 of the second heavy chain under b) the aminoacid at position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E) or aspartic acid (D) (numberingaccording to Kabat EU index).

In one aspect, in the constant domain CL of the second heavy chain theamino acids at position 124 and 123 are substituted by K (numberingaccording to Kabat EU index).

In one aspect, in the constant domain CL of the second heavy chain theamino acid at position 123 is substituted by R and the amino acid asposition 124 is substituted by K (numbering according to Kabat EUindex).

In one aspect, in the constant domain CH1 of the second light chain theamino acids at position 147 and 213 are substituted by E (numberingaccording to EU index of Kabat).

In one aspect, in the constant domain CL of the first light chain theamino acids at position 124 and 123 are substituted by K, and in theconstant domain CH1 of the first heavy chain the amino acids at position147 and 213 are substituted by E (numbering according to Kabat EUindex).

In one aspect, in the constant domain CL of the first light chain theamino acid at position 123 is substituted by R and the amino acid atposition 124 is substituted by K, and in the constant domain CH1 of thefirst heavy chain the amino acids at position 147 and 213 are bothsubstituted by E (numbering according to Kabat EU index).

In one aspect, in the constant domain CL of the second heavy chain theamino acids at position 124 and 123 are substituted by K. and wherein inthe constant domain CH1 of the second light chain the amino acids atposition 147 and 213 are substituted by E, and in the variable domain VLof the first light chain the amino acid at position 38 is substituted byK, in the variable domain VH of the first heavy chain the amino acid atposition 39 is substituted by E, in the variable domain VL of the secondheavy chain the amino acid at position 38 is substituted by K, and inthe variable domain VH of the second light chain the amino acid atposition 39 is substituted by E (numbering according to Kabat EU index).

In one aspect, the bispecific antibody is a bivalent antibody comprising

-   -   a) a first light chain and a first heavy chain of an antibody        specifically binding to a first antigen, and    -   b) a second light chain and a second heavy chain of an antibody        specifically binding to a second antigen, wherein the variable        domains VL and VH of the second light chain and the second heavy        chain are replaced by each other, and wherein the constant        domains CL and CH1 of the second light chain and the second        heavy chain are replaced by each other.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain und a) are isolated chains.In the antibody under b) within the light chain the variable light chaindomain VL is replaced by the variable heavy chain domain VH of saidantibody, and the constant light chain domain CL is replaced by theconstant heavy chain domain CH1 of said antibody; and within the heavychain the variable heavy chain domain VH is replaced by the variablelight chain domain VL of said antibody, and the constant heavy chaindomain CH1 is replaced by the constant light chain domain CL of saidantibody.

In one aspect, the bispecific antibody is a bivalent antibody comprising

-   -   a) a first light chain and a first heavy chain of an antibody        specifically binding to a first antigen, and    -   b) a second light chain and a second heavy chain of an antibody        specifically binding to a second antigen, wherein the constant        domains CL and CH1 of the second light chain and the second        heavy chain are replaced by each other.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain under a) are isolated chains.In the antibody under b) within the light chain the constant light chaindomain CL is replaced by the constant heavy chain domain CH1 of saidantibody; and within the heavy chain the constant heavy chain domain CH1is replaced by the constant light chain domain CL of said antibody.

In one aspect, the bispecific antibody is a bispecific antibodycomprising

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains, and    -   b) one, two, three or four single chain Fab fragments        specifically binding to a second antigen,    -   wherein said single chain Fab fragments under b) are fused to        said full length antibody under a) via a peptide linker at the        C- or N-terminus of the heavy or light chain of said full length        antibody.

In one aspect, one or two identical single chain Fab fragments bindingto a second antigen are fused to the full length antibody via a peptidelinker at the C terminus of the heavy or light chains of said fulllength antibody.

In one aspect, one or two identical single chain Fab (scFab) fragmentsbinding to a second antigen are fused to the full length antibody via apeptide linker at the C terminus of the heavy chains of said full lengthantibody.

In one aspect, one or two identical single chain Fab (scFab) fragmentsbinding to a second antigen are fused to the full length antibody via apeptide linker at the C terminus of the light chains of said full lengthantibody.

In one aspect, two identical single chain Fab (scFab) fragments bindingto a second antigen are fused to the full length antibody via a peptidelinker at the C-terminus of each heavy or light chain of said fulllength antibody.

In one aspect, two identical single chain Fab (scFab) fragments bindingto a second antigen are fused to the full length antibody via a peptidelinker at the C-terminus of each heavy chain of said full lengthantibody.

In one aspect, two identical single chain Fab (scFab) fragments bindingto a second antigen are fused to the full length antibody via a peptidelinker at the C-terminus of each light chain of said full lengthantibody.

In one aspect, the bispecific antibody is a trivalent antibodycomprising

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains,    -   b) a first polypeptide consisting of        -   ba) an antibody heavy chain variable domain (VH), or        -   bb) an antibody heavy chain variable domain (VH) and an            antibody constant domain 1 (CH1),    -   wherein said first polypeptide is fused with the N-terminus of        its VH domain via a peptidic linker to the C-terminus of one of        the two heavy chains of said full length antibody,    -   c) a second polypeptide consisting of        -   ca) an antibody light chain variable domain (VL), or        -   cb) an antibody light chain variable domain (VL) and an            antibody light chain constant domain (CL),    -   wherein said second polypeptide is fused with the N-terminus of        the VL domain via a peptide linker to the C-terminus of the        other of the two heavy chains of said full length antibody, and    -   wherein the antibody heavy chain variable domain (VH) of the        first polypeptide and the antibody light chain variable domain        (VL) of the second polypeptide together form an antigen binding        domain specifically binding to a second antigen.

In one aspect, the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) are linked and stabilized via an interchaindisulfide bridge by introduction of a disulfide bond between thefollowing positions:

-   -   (i) heavy chain variable domain position 44 to light chain        variable domain position 100, or    -   (ii) heavy chain variable domain position 105 to light chain        variable domain position 43, or    -   (iii) heavy chain variable domain position 101 to light chain        variable domain position 100 (numbering always according to        Kabat EU index).

Techniques to introduce unnatural disulfide bridges for stabilizationare described e.g. in WO 94/029350, Rajagopal, V., et al., Prot. Eng.(1997) 1453-1459; Kobayashi, H., et al., Nucl. Med. Biol. 25 (1998)387-393; and Schmidt, M., et al., Oncogene 18 (1999) 1711-1721. In oneembodiment the optional disulfide bond between the variable domains ofthe polypeptides under b) and c) is between heavy chain variable domainposition 44 and light chain variable domain position 100. In oneembodiment the optional disulfide bond between the variable domains ofthe polypeptides under b) and c) is between heavy chain variable domainposition 105 and light chain variable domain position 43 (numberingalways according to Kabat). In one embodiment a trivalent, bispecificantibody without said optional disulfide stabilization between thevariable domains VH and VL of the single chain Fab fragments ispreferred.

In one aspect, the bispecific antibody is a trispecific or tetraspecificantibody, comprising

-   -   a) a first light chain and a first heavy chain of a full length        antibody which specifically binds to a first antigen, and    -   b) a second (modified) light chain and a second (modified) heavy        chain of a full length antibody which specifically binds to a        second antigen, wherein the variable domains VL and VH are        replaced by each other, and/or wherein the constant domains CL        and CH1 are replaced by each other, and    -   c) wherein one to four antigen binding domains which        specifically bind to one or two further antigens (i.e. to a        third and/or fourth antigen) are fused via a peptide linker to        the C- or N-terminus of the light chains or heavy chains of a)        and/or b).

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain und a) are isolated chains.

In one aspect, the trispecific or tetraspecific antibody comprises underc) one or two antigen binding domains which specifically bind to one ortwo further antigens.

In one aspect, the antigen binding domains are selected from the groupof a scFv fragment and a scFab fragment.

In one aspect, the antigen binding domains are scFv fragments.

In one aspect, the antigen binding domains are scFab fragments.

In one aspect, the antigen binding domains are fused to the C-terminusof the heavy chains of a) and/or b).

In one aspect, the trispecific or tetraspecific antibody comprises underc) one or two antigen binding domains which specifically bind to onefurther antigen.

In one aspect, the trispecific or tetraspecific antibody comprises underc) two identical antigen binding domains which specifically bind to athird antigen. In one preferred embodiment such two identical antigenbinding domains are fused both via the same peptidic linker to theC-terminus of the heavy chains of a) and b). In one preferred embodimentthe two identical antigen binding domains are either a scFv fragment ora scFab fragment.

In one aspect, the trispecific or tetraspecific antibody comprises underc) two antigen binding domains which specifically bind to a third and afourth antigen. In one embodiment said two antigen binding domains arefused both via the same peptide connector to the C-terminus of the heavychains of a) and b). In one preferred embodiment said two antigenbinding domains are either a scFv fragment or a scFab fragment.

In one aspect, the bispecific antibody is a bispecific, tetravalentantibody comprising

-   -   a) two light chains and two heavy chains of an antibody, which        specifically bind to a first antigen (and comprise two Fab        fragments),    -   b) two additional Fab fragments of an antibody, which        specifically bind to a second antigen, wherein said additional        Fab fragments are fused both via a peptidic linker either to the        C- or N-termini of the heavy chains of a), and    -   wherein in the Fab fragments the following modifications were        performed    -   (i) in both Fab fragments of a), or in both Fab fragments of b),        the variable domains VL and VH are replaced by each other,        and/or the constant domains CL and CH1 are replaced by each        other, or    -   (ii) in both Fab fragments of a) the variable domains VL and VH        are replaced by each other, and the constant domains CL and CH1        are replaced by each other, and in both Fab fragments of b) the        variable domains VL and VH are replaced by each other, or the        constant domains CL and CH1 are replaced by each other, or    -   (iii) in both Fab fragments of a) the variable domains VL and VH        are replaced by each other, or the constant domains CL and CH1        are replaced by each other, and in both Fab fragments of b) the        variable domains VL and VH are replaced by each other, and the        constant domains CL and CH1 are replaced by each other, or    -   (iv) in both Fab fragments of a) the variable domains VL and VH        are replaced by each other, and in both Fab fragments of b) the        constant domains CL and CH1 are replaced by each other, or    -   (v) in both Fab fragments of a) the constant domains CL and CH1        are replaced by each other, and in both Fab fragments of b) the        variable domains VL and VH are replaced by each other.

In one aspect, said additional Fab fragments are fused both via apeptidic linker either to the C-termini of the heavy chains of a), or tothe N-termini of the heavy chains of a).

In one aspect, said additional Fab fragments are fused both via apeptidic linker either to the C-termini of the heavy chains of a).

In one aspect, said additional Fab fragments are fused both via apeptide linker to the N-termini of the heavy chains of a).

In one aspect, in the Fab fragments the following modifications areperformed: in both Fab fragments of a), or in both Fab fragments of b),the variable domains VL and VH are replaced by each other, and/or theconstant domains CL and CH1 are replaced by each other.

In one aspect, the bispecific antibody is a tetravalent antibodycomprising:

-   -   a) a (modified) heavy chain of a first antibody, which        specifically binds to a first antigen and comprises a first        VH-CH1 domain pair, wherein to the C terminus of said heavy        chain the N-terminus of a second VH-CH1 domain pair of said        first antibody is fused via a peptide linker,    -   b) two light chains of said first antibody of a),    -   c) a (modified) heavy chain of a second antibody, which        specifically binds to a second antigen and comprises a first        VH-CL domain pair, wherein to the C-terminus of said heavy chain        the N-terminus of a second VH-CL domain pair of said second        antibody is fused via a peptide linker, and    -   d) two (modified) light chains of said second antibody of c),        each comprising a CL-CH1 domain pair.

In one aspect, the bispecific antibody comprises

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to a first antigen, and    -   b) the heavy chain and the light chain of a second full length        antibody that specifically binds to a second antigen, wherein        the N-terminus of the heavy chain is connected to the C-terminus        of the light chain via a peptide linker.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain are isolated chains.

In one aspect, the bispecific antibody comprises

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains, and    -   b) an Fv fragment specifically binding to a second antigen        comprising a VH2 domain and a VL2 domain, wherein both domains        are connected to each other via a disulfide bridge,    -   wherein only either the VH2 domain or the VL2 domain is fused        via a peptide linker to the heavy or light chain of the full        length antibody specifically binding to a first antigen.

In the bispecific antibody the heavy chains and the light chains undera) are isolated chains.

In one aspect, the other of the VH2 domain or the VL2 domain is notfused via a peptide linker to the heavy or light chain of the fulllength antibody specifically binding to a first antigen.

In all aspects as reported herein the first light chain comprises a VLdomain and a CL domain and the first heavy chain comprises a VH domain,a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

In one aspect, the bispecific antibody is a trivalent antibodycomprising

-   -   a) two Fab fragments that specifically binds to a first antigen,    -   b) one CrossFab fragment that specifically binds to a second        antigen in which the CH1 and the CL domain are exchanged for        each other,    -   c) one Fc-region comprising a first Fc-region heavy chain and a        second Fc region heavy chain,    -   wherein the C-terminus of CH1 domains of the two Fab fragments        are connected to the N-terminus of the heavy chain Fc-region        polypeptides, and wherein the C-terminus of the CL domain of the        CrossFab fragment is connected to the N-terminus of the VH        domain of one of the Fab fragments.

In one aspect, the bispecific antibody is a trivalent antibodycomprising

-   -   a) two Fab fragments that specifically binds to a first antigen,    -   b) one CrossFab fragment that specifically binds to a second        antigen in which the CH1 and the CL domain are exchanged for        each other,    -   c) one Fc-region comprising a first Fc-region heavy chain and a        second Fc region heavy chain,    -   wherein the C-terminus of CH1 domain of the first Fab fragment        is connected to the N-terminus of one of the heavy chain        Fc-region polypeptides and the C-terminus of the CL-domain of        the CrossFab fragment is connected to the N-terminus of the        other heavy chain Fc-region polypeptide, and wherein the        C-terminus of the CH1 domain of the second Fab fragment is        connected to the N-terminus of the VH domain of the first Fab        fragment or to the N-terminus of the VH domain of the CrossFab        fragment.

In one aspect, the bispecific antibody comprises

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains, and    -   b) a Fab fragment specifically binding to a second antigen        comprising a VH2 domain and a VL2 domain comprising a heavy        chain fragment and a light chain fragment, wherein within the        light chain fragment the variable light chain domain VL2 is        replaced by the variable heavy chain domain VH2 of said        antibody, and within the heavy chain fragment the variable heavy        chain domain VH2 is replaced by the variable light chain domain        VL2 of said antibody    -   wherein the heavy chain Fab fragment is inserted between the CH1        domain of one of the heavy chains of the full length antibody        and the respective Fc-region of the full length antibody, and        the N-terminus of the light chain Fab fragment is conjugated to        the C-terminus of the light chain of the full length antibody        that is paired with the heavy chain of the full length antibody        into which the heavy chain Fab fragment has been inserted.

In one aspect, the bispecific antibody comprises

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains, and    -   b) a Fab fragment specifically binding to a second antigen        comprising a VH2 domain and a VL2 domain comprising a heavy        chain fragment and a light chain fragment, wherein within the        light chain fragment the variable light chain domain VL2 is        replaced by the variable heavy chain domain VH2 of said        antibody, and within the heavy chain fragment the variable heavy        chain domain VH2 is replaced by the variable light chain domain        VL2 of said antibody and wherein the C-terminus of the heavy        chain fragment of the Fab fragment is conjugated to the        N-terminus of one of the heavy chains of the full length        antibody and the C-terminus of the light chain fragment of the        Fab fragment is conjugated to the N-terminus of the light chain        of the full length antibody that pairs with the heavy chain of        the full length antibody to which the heavy chain fragment of        the Fab fragment is conjugated.

Polynucleotides

The invention further provides isolated polynucleotides encoding abispecific antibody as described herein or a fragment thereof.

The term “nucleic acid molecule” or “polynucleotide” includes anycompound and/or substance that comprises a polymer of nucleotides. Eachnucleotide is composed of a base, specifically a purine- or pyrimidinebase (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil(U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group.Often, the nucleic acid molecule is described by the sequence of bases,whereby said bases represent the primary structure (linear structure) ofa nucleic acid molecule. The sequence of bases is typically representedfrom 5′ to 3′. Herein, the term nucleic acid molecule encompassesdeoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) andgenomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA),synthetic forms of DNA or RNA, and mixed polymers comprising two or moreof these molecules. The nucleic acid molecule may be linear or circular.In addition, the term nucleic acid molecule includes both, sense andantisense strands, as well as single stranded and double stranded forms.Moreover, the herein described nucleic acid molecule can containnaturally occurring or non-naturally occurring nucleotides. Examples ofnon-naturally occurring nucleotides include modified nucleotide baseswith derivatized sugars or phosphate backbone linkages or chemicallymodified residues. Nucleic acid molecules also encompass DNA and RNAmolecules which are suitable as a vector for direct expression of anantibody of the invention in vitro and/or in vivo, e.g., in a host orpatient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can beunmodified or modified. For example, mRNA can be chemically modified toenhance the stability of the RNA vector and/or expression of the encodedmolecule so that mRNA can be injected into a subject to generate theantibody in vivo (see e.g., Stadler et al, Nature Medicine 2017,published online 12 Jun. 2017, doi:10.1038/nm.4356 or EP 2 101 823 B1).

An “isolated” polynucleotide refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatedpolynucleotide includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

The isolated polynucleotides encoding bispecific antibodies of theinvention may be expressed as a single polynucleotide that encodes theentire antigen binding molecule or as multiple (e.g., two or more)polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional antigen bindingmolecule. For example, the light chain portion of an immunoglobulin maybe encoded by a separate polynucleotide from the heavy chain portion ofthe immunoglobulin. When co-expressed, the heavy chain polypeptides willassociate with the light chain polypeptides to form the immunoglobulin.

In some aspects, the isolated polynucleotide encodes a polypeptidecomprised in the bispecific antibody according to the invention asdescribed herein.

In one aspect, the present invention is directed to isolatedpolynucleotides encoding a bispecific antibody comprising a firstantigen binding domain that specifically binds to programmed cell deathprotein 1 (PD1) and a second antigen binding domain that specificallybinds to Lymphocyte activation gene-3 (LAG3), wherein said first antigenbinding domain specifically binding to PD1 comprises a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and(iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:3; and a VLdomain comprising (i) HVR-L1 comprising the amino acid sequence of SEQID NO:4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:6.

B. Recombinant Methods

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. For these methods one ormore isolated nucleic acid(s) encoding an antibody are provided.

In case of a native antibody or native antibody fragment two nucleicacids are required, one for the light chain or a fragment thereof andone for the heavy chain or a fragment thereof. Such nucleic acid(s)encode an amino acid sequence comprising the VL and/or an amino acidsequence comprising the VH of the antibody (e.g., the light and/or heavychain(s) of the antibody). These nucleic acids can be on the sameexpression vector or on different expression vectors. In case of certainbispecific antibodies with heterodimeric heavy chains four nucleic acidsare required, one for the first light chain, one for the first heavychain comprising the first heteromonomeric Fc-region polypeptide, onefor the second light chain, and one for the second heavy chaincomprising the second heteromonomeric Fc-region polypeptide. The fournucleic acids can be comprised in one or more nucleic acid molecules orexpression vectors. For example, such nucleic acid(s) encode an aminoacid sequence comprising the first VL and/or an amino acid sequencecomprising the first VH including the first heteromonomeric Fc-regionand/or an amino acid sequence comprising the second VL and/or an aminoacid sequence comprising the second VH including the secondheteromonomeric Fc-region of the antibody (e.g., the first and/or secondlight and/or the first and/or second heavy chains of the antibody).These nucleic acids can be on the same expression vector or on differentexpression vectors, normally these nucleic acids are located on two orthree expression vectors, i.e. one vector can comprise more than one ofthese nucleic acids. Examples of these bispecific antibodies areCrossMabs and T-cell bispecifics (see, e.g. Schaefer. W. et al, PNAS,108 (2011) 11187-1191). For example, one of the heteromonomeric heavychain comprises the so-called “knob mutations” (T366W and optionally oneof S354C or Y349C) and the other comprises the so-called “holemutations” (T366S, L368A and Y407V and optionally Y349C or S354C) (see,e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73).

In one aspect, isolated nucleic acid encoding a bispecific antibodydescribed herein is provided. Such nucleic acid may encode an amino acidsequence comprising the VL and/or an amino acid sequence comprising theVH of the antigen binding domains that specifically bind to PD1 andLAG3, respectively (e.g., in the light and/or heavy chains of theantibody). In a further aspect, one or more vectors (e.g., expressionvectors) comprising such nucleic acid are provided. In a further aspect,a host cell comprising such nucleic acid is provided. In one suchaspect, a host cell comprises (e.g., has been transformed with): (1) afirst vector comprising a first pair of nucleic acids that encode aminoacid sequences one of them comprising the first VL and the othercomprising the first VH of the antibody and a second vector comprising asecond pair of nucleic acids that encode amino acid sequences one ofthem comprising the second VL and the other comprising the second VH ofthe antibody, or (2) a first vector comprising a first nucleic acid thatencode an amino acid sequence comprising one of the variable domains(preferably a light chain variable domain), a second vector comprising apair of nucleic acids that encode amino acid sequences one of themcomprising a light chain variable domain and the other comprising thefirst heavy chain variable domain, and a third vector comprising a pairof nucleic acids that encode amino acid sequences one of them comprisingthe respective other light chain variable domain as in the second vectorand the other comprising the second heavy chain variable domain, or (3)a first vector comprising a nucleic acid that encodes an amino acidsequence comprising the first VL of the antibody, a second vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe first VH of the antibody, a third vector comprising a nucleic acidthat encodes an amino acid sequence comprising the second VL of theantibody, and a fourth vector comprising a nucleic acid that encodes anamino acid sequence comprising the second VH of the antibody. In oneaspect, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO)cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one aspect, amethod of making a bispecific antibody is provided, wherein the methodcomprises culturing a host cell comprising a nucleic acid encoding theantibody, as provided above, under conditions suitable for expression ofthe antibody, and optionally recovering the antibody from the host cell(or host cell culture medium).

For recombinant production of the bispecific antibodies comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein, nucleic acid encoding the bispecific antibodies, e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such nucleic acid maybe readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.No. 5,648,237, U.S. Pat. No. 5,789,199, and U.S. Pat. No. 5,840,523.(See also Charlton, K. A., In: Methods in Molecular Biology, Vol. 248,Lo, B. K. C. (ed.), Humana Press, Totowa, N.J. (2003), pp. 245-254,describing expression of antibody fragments in E. coli.) Afterexpression, the antibody may be isolated from the bacterial cell pastein a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibodies arealso derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains have been identified which may beused in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV 1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV 1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR−CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

C. Assays

The bispecific antibodies comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 provided herein may be identified, screenedfor, or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Affinity Assays

The affinity of the bispecific antigen binding molecules, antibodies andantibody fragments provided herein for the corresponding antigens can bedetermined in accordance with the methods set forth in the Examples bysurface plasmon resonance (SPR), using standard instrumentation such asa Biacore® instrument (GE Healthcare), and receptors or target proteinssuch as may be obtained by recombinant expression. A specificillustrative and exemplary embodiment for measuring binding affinity isdescribed in Examples 2, 8 or 11. According to one aspect, K_(D) ismeasured by surface plasmon resonance using a BIACORE® T100 machine (GEHealthcare) at 25° C.

2. Binding Assays and Other Assays

In one aspect, the bispecific antibodies of the invention are tested forits antigen binding activity, e.g., by known methods such as ELISA,Western blot, etc. Binding of the bispecific antibodies provided hereinto the corresponding recombinant antigen or to antigen-expressing cellsmay be evaluated by ELISA as described in Examples 8 or 11.

In a further aspect, fresh peripheral blood mononuclear cells (PBMCs)are used in binding assays to show binding to different peripheral bloodmononuclear cells (PBMC) such as monocytes, NK cells and T cells.

In another aspect, a cellular dimerization assay was used to demonstratethe dimerization or at last binding/interaction of two differentreceptors PD1 and LAG3, which are cytosolically fused with two fragmentsof an enzyme, upon ligation or cross-linking with a bispecific antibodyagainst both targets. Hereby only one receptor alone shows no enzymaticactivity. For this specific interaction, the cytosolic C-terminal endsof both receptors were individually fused to heterologous subunits of areporter enzym. A single enzyme subunit alone showed no reporteractivity. However, simultaneous binding to both receptors was expectedto lead to local cytocolic accumulation of both receptors,complementation of the two heterologous enzyme subunits, and finally toresult in the formation of a specific and functional enzyme thathydrolyzes a substrate thereby generating a chemiluminescent signal(Example 11).

3. Activity Assays

In one aspect, assays are provided for identifying a bispecific antibodycomprising a first antigen binding domain that specifically binds to PD1and a second antigen binding domain that specifically binds to LAG3having biological activity. Biological activity may include, e.g., theability to enhance the activation and/or proliferation of differentimmune cells, especially T-cells, secretion of immune-modulatingcytokines such IFNγ or TNF-alpha, blocking the PD1 pathway, blocking theLAG3 pathway, killing of tumor cells. Antibodies having such biologicalactivity in vivo and/or in vitro are also provided.

In certain aspects, an antibody of the invention is tested for suchbiological activity. In one aspect, provided is an immune cell assaywhich measures the activation of lymphocytes from one individual (donorX) to lymphocytes from another individual (donor Y). The mixedlymphocyte reaction (MLR) can demonstrate the effect of blocking the PD1pathway to lymphocyte effector cells. T cells in the assay were testedfor activation and their IFN-gamma secretion in the presence or absenceof bispecific antibodies of the invention. The assay is described inmore detail in Example 9.

D. Immunoconjugates

The invention also provides immunoconjugates comprising a bispecificantibody of the invention conjugated to one or more cytotoxic agents,such as chemotherapeutic agents or drugs, growth inhibitory agents,toxins (e.g., protein toxins, enzymatically active toxins of bacterial,fungal, plant, or animal origin, or fragments thereof), or radioactiveisotopes.

E. Methods and Compositions for Diagnostics and Detection

In certain aspects, any of the bispecific antibodies comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 provided hereinmay be useful for detecting the presence of both PD1 and LAG3 in abiological sample. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue, such as AML stem cancercells.

In one aspect, a bispecific antibody for use in a method of diagnosis ordetection is provided. In a further aspect, a method of detecting thepresence of both PD1 and LAG3 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with a bispecific antibody as described herein under conditionspermissive for binding of the bispecific antibody to both PD1 and LAG3,and detecting whether a complex is formed between the bispecificantibody and both antigens. Such method may be an in vitro or in vivomethod. In one embodiment, the bispecific antibody is used to selectsubjects eligible for therapy with a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 antibody, e.g.where PD1 and LAG3 are biomarkers for selection of patients.

In certain aspects, labeled bispecific antibodies are provided. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes 32P, 14C, 125I, 3H, and 131I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels or stable free radicals.

F. Pharmaceutical Compositions, Formulations and Routes of Administation

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the bispecific antibodies comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 provided herein, e.g.,for use in any of the below therapeutic methods. In one embodiment, apharmaceutical composition comprises any of the bispecific antibodiesprovided herein and at least one pharmaceutically acceptable excipient.In another embodiment, a pharmaceutical composition comprises any of thebispecific antibodies provided herein and at least one additionaltherapeutic agent, e.g., as described below.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more bispecific antibodiesdissolved or dispersed in a pharmaceutically acceptable excipient. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that are generally non-toxic torecipients at the dosages and concentrations employed, i.e. do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The preparation of a pharmaceutical composition thatcontains at least one bispecific antibody and optionally an additionalactive ingredient will be known to those of skill in the art in light ofthe present disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. In particular, the compositions are lyophilized formulationsor aqueous solutions. As used herein, “pharmaceutically acceptableexcipient” includes any and all solvents, buffers, dispersion media,coatings, surfactants, antioxidants, preservatives (e.g. antibacterialagents, antifungal agents), isotonic agents, salts, stabilizers andcombinations thereof, as would be known to one of ordinary skill in theart.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the bispecific antibodies of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiological saline buffer. The solution may contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Alternatively,the bispecific antibodies may be in powder form for constitution with asuitable vehicle, e.g., sterile pyrogen-free water, before use. Sterileinjectable solutions are prepared by incorporating the fusion proteinsof the invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated below, as required.Sterility may be readily accomplished. e.g., by filtration throughsterile filtration membranes. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The composition must be stable under theconditions of manufacture and storage, and preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Itwill be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less that 0.5 ng/mg protein.Suitable pharmaceutically acceptable excipients include, but are notlimited to: buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

Exemplary pharmaceutically acceptable excipients herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958.

Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

In addition to the compositions described previously, the bispecificantibodies may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection.

Thus, for example, the bispecific antibodies may be formulated withsuitable polymeric or hydrophobic materials (for example as emulsion inan acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the bispecific antibodies of theinvention may be manufactured by means of conventional mixing,dissolving, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmaceutical compositions may be formulated in conventionalmanner using one or more physiologically acceptable carriers, diluents,excipients or auxiliaries which facilitate processing of the proteinsinto preparations that can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

The bispecific antibodies may be formulated into a composition in a freeacid or base, neutral or salt form. Pharmaceutically acceptable saltsare salts that substantially retain the biological activity of the freeacid or base. These include the acid addition salts, e.g. those formedwith the free amino groups of a proteinaceous composition, or which areformed with inorganic acids such as for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric ormandelic acid. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine. Pharmaceuticalsalts tend to be more soluble in aqueous and other protic solvents thanare the corresponding free base forms.

The composition herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the bispecific antibodies comprising a first antigen bindingdomain that specifically binds to PD1 and a second antigen bindingdomain that specifically binds to LAG3 provided herein may be used intherapeutic methods.

For use in therapeutic methods, bispecific antibodies comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as defined hereinbefore can be formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners.

In one aspect, bispecific antibodies comprising a first antigen bindingdomain that specifically binds to PD1 and a second antigen bindingdomain that specifically binds to LAG3 as defined herein for use as amedicament are provided. In further aspects, bispecific antibodiescomprising a first antigen binding domain that specifically binds to PD1and a second antigen binding domain that specifically binds to LAG3 asdefined herein for use in treating a disease, in particular for use inthe treatment of cancer, are provided. In certain embodiments,bispecific antibodies comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 for use in a method of treatment areprovided. In one embodiment, the invention provides bispecificantibodies comprising a first antigen binding domain that specificallybinds to PD1 and a second antigen binding domain that specifically bindsto LAG3 as described herein for use in the treatment of a disease in anindividual in need thereof. In certain embodiments, the inventionprovides bispecific antibodies comprising a first antigen binding domainthat specifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 for use in a method of treating an individualhaving a disease comprising administering to the individual atherapeutically effective amount of the bispecific antibody. In certainembodiments the disease to be treated is cancer. In another aspect, thedisease to be treated is an infectious disease, in particular a chronicviral infection like HIV (human immunodeficiency virus), HBV (hepatitisB virus), HCV (hepatitis C), HSV1 (herpes simplex virus type 1), CMV(cytomegalovirus), LCMV (lymphocytic chroriomeningitis virus) or EBV(Epstein-Barr virus). The subject, patient, or “individual” in need oftreatment is typically a mammal, more specifically a human.

In a further aspect, the invention provides for the use of bispecificantibodies comprising a first antigen binding domain that specificallybinds to PD1 and a second antigen binding domain that specifically bindsto LAG3 as defined herein before in the manufacture or preparation of amedicament for the treatment of a disease in an individual in needthereof. In one embodiment, the medicament is for use in a method oftreating a disease comprising administering to an individual having thedisease a therapeutically effective amount of the medicament.

In certain aspects, the disease to be treated is a proliferativedisorder, particularly cancer. Examples of cancers include bladdercancer, brain cancer, head and neck cancer, pancreatic cancer, lungcancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer,endometrial cancer, esophageal cancer, colon cancer, colorectal cancer,rectal cancer, gastric cancer, prostate cancer, blood cancer, skincancer, squamous cell carcinoma, bone cancer, and kidney cancer. Othercell proliferation disorders that can be treated using bispecificantibodies comprising a first antigen binding domain that specificallybinds to PD1 and a second antigen binding domain that specifically bindsto LAG3 according to the invention include, but are not limited toneoplasms located in the abdomen, bone, breast, digestive system, liver,pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous system(central and peripheral), lymphatic system, pelvic, skin, soft tissue,spleen, thoracic region, and urogenital system. Also included arepre-cancerous conditions or lesions and cancer metastases. In certainaspects, the cancer is chosen from the group consisting of renal cellcancer, skin cancer, lung cancer, colorectal cancer, breast cancer,brain cancer, head and neck cancer. In further aspects, the cancer ischosen from carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin'slymphoma), blastoma, sarcoma, and leukemia. In another aspect, thecancer is to be treated is selected from squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, leukemia and other lymphoproliferative disorders, and varioustypes of head and neck cancer.

In a further aspect, the disease to be treated is an infectious disease,in particular a chronic viral infection. The term “chronic viralinfection” refers to a subject afflicted or infected with a chronicvirus. Examples for chronic viral infections are human immunodeficiencyvirus (HIV), hepatitis B viral infection (HBV), hepatitis C viralinfection (HCV), herpes simplex virus 1 (HSV1), cytomegalovirus (CMV),lymphocytic choriomeningitis virus (LCMV) or Epstein-Barr virus (EBV).

A skilled artisan readily recognizes that in many cases the bispecificmolecule may not provide a cure but may only provide partial benefit. Insome embodiments, a physiological change having some benefit is alsoconsidered therapeutically beneficial. Thus, in some embodiments, anamount of the bispecific antibody that provides a physiological changeis considered an “effective amount” or a “therapeutically effectiveamount”.

In a further aspect, the invention provides a method for treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of bispecific antibodies comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 of the invention.In one embodiment a composition is administered to said individual,comprising a bispecific antibody of the invention in a pharmaceuticallyacceptable form. In certain embodiments the disease to be treated is aproliferative disorder. In a particular embodiment the disease iscancer. In certain embodiments the method further comprisesadministering to the individual a therapeutically effective amount of atleast one additional therapeutic agent, e.g. an anti-cancer agent if thedisease to be treated is cancer. In another aspect, the disease is achronic viral infection. An “individual” according to any of the aboveembodiments may be a mammal, preferably a human.

For the prevention or treatment of disease, the appropriate dosage of abispecific antibodies comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 of the invention (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the route ofadministration, the body weight of the patient, the type of fusionprotein, the severity and course of the disease, whether the bispecificantibody is administered for preventive or therapeutic purposes,previous or concurrent therapeutic interventions, the patient's clinicalhistory and response to the fusion protein, and the discretion of theattending physician. The practitioner responsible for administrationwill, in any event, determine the concentration of active ingredient(s)in a composition and appropriate dose(s) for the individual subject.Various dosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

The bispecific antibody comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 as defined herein is suitably administered tothe patient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1mg/kg-10 mg/kg) of the bispecific antibody can be an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. One typicaldaily dosage might range from about 1 μg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentwould generally be sustained until a desired suppression of diseasesymptoms occurs. One exemplary dosage of the bispecific antibody wouldbe in the range from about 0.005 mg/kg to about 10 mg/kg. In otherexamples, a dose may also comprise from about 1 μg/kg body weight, about5 μg/kg body weight, about 10 μg/kg body weight, about 50 μg/kg bodyweight, about 100 μg/kg body weight, about 200 μg/kg body weight, about350 μg/kg body weight, about 500 μg/kg body weight, about 1 mg/kg bodyweight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg bodyweight, about 350 mg/kg body weight, about 500 mg/kg body weight, toabout 1000 mg/kg body weight or more per administration, and any rangederivable therein. In examples of a derivable range from the numberslisted herein, a range of about 5 mg/kg body weight to about 100 mg/kgbody weight, about 5 μg/kg body weight to about 500 mg/kg body weightetc., can be administered, based on the numbers described above. Thus,one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg(or any combination thereof) may be administered to the patient. Suchdoses may be administered intermittently, e.g. every week or every threeweeks (e.g. such that the patient receives from about two to abouttwenty, or e.g. about six doses of the fusion protein). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The bispecific antibodies comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 as defined herein will generally be used inan amount effective to achieve the intended purpose. For use to treat orprevent a disease condition, the bispecific antibodies of the invention,or pharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the bispecific antibody which are sufficient tomaintain therapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 50 mg/kg/day, typically from about 0.5to 1 mg/kg/day. Therapeutically effective plasma levels may be achievedby administering multiple doses each day. Levels in plasma may bemeasured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the bispecific antibody may not be related toplasma concentration. One skilled in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the bispecific antibodies describedherein will generally provide therapeutic benefit without causingsubstantial toxicity. Toxicity and therapeutic efficacy of a fusionprotein can be determined by standard pharmaceutical procedures in cellculture or experimental animals. Cell culture assays and animal studiescan be used to determine the LD₅₀ (the dose lethal to 50% of apopulation) and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀.Bispecific antibodies that exhibit large therapeutic indices arepreferred. In one embodiment, the bispecific antibody according to thepresent invention exhibits a high therapeutic index. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosages suitable for use in humans. The dosage lies preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage may vary within this range dependingupon a variety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with bispecific antibodiesof the invention would know how and when to terminate, interrupt, oradjust administration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Other Agents and Treatments

The bispecific antibodies comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 as described herein before may beadministered in combination with one or more other agents in therapy.For instance, a bispecific antibody of the invention may beco-administered with at least one additional therapeutic agent. The term“therapeutic agent” encompasses any agent that can be administered fortreating a symptom or disease in an individual in need of suchtreatment. Such additional therapeutic agent may comprise any activeingredients suitable for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. In certain embodiments, an additional therapeuticagent is another anti-cancer agent.

In one aspect of the invention, the bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein or a pharmaceutical composition comprising said bispecificantibody is for use in the prevention or treatment of cancer, whereinthe bispecific antibody is administered in combination with achemotherapeutic agent, radiation and/or other agents for use in cancerimmunotherapy.

In a particular aspect of the invention, the bispecific antibodycomprising a first antigen binding domain that specifically binds to PD1and a second antigen binding domain that specifically binds to LAG3 asdescribed herein or a pharmaceutical composition comprising saidbispecific antibody is for use in the prevention or treatment of cancer,wherein the bispecific antibody is administered in combination with anT-cell activating anti-CD3 bispecific antibody, in particular ananti-CEA/anti-CD3 bispecific antibody. In one aspect, theanti-CEA/anti-CD3 bispecific antibody is a T-cell activating anti-CD3bispecific antibody comprising a second antigen binding domaincomprising (a) a heavy chain variable region (V_(H)CEA) comprisingCDR-H1 sequence of SEQ ID NO: 154, CDR-H2 sequence of SEQ ID NO:155, andCDR-H3 sequence of SEQ ID NO: 156, and/or a light chain variable region(V_(L)CEA) comprising CDR-L1 sequence of SEQ ID NO:157, CDR-L2 sequenceof SEQ ID NO:158, and CDR-L3 sequence of SEQ ID NO: 159, or (b) a heavychain variable region (V_(H)CEA) comprising CDR-H1 sequence of SEQ IDNO:162, CDR-H2 sequence of SEQ ID NO: 163, and CDR-H3 sequence of SEQ IDNO: 164, and/or a light chain variable region (V_(L)CEA) comprisingCDR-L1 sequence of SEQ ID NO: 165, CDR-L2 sequence of SEQ ID NO: 166,and CDR-L3 sequence of SEQ ID NO: 167. In one aspect, theanti-CEA/anti-CD3 bispecific antibody is a T-cell activating anti-CD3bispecific antibody comprising a heavy chain variable region (V_(H)CEA)comprising the amino acid sequence of SEQ ID NO: 160 and/or a lightchain variable region (V_(L)CEA) comprising the amino acid sequence ofSEQ ID NO: 161 or a second antigen binding domain comprising a heavychain variable region (V_(H)CEA) comprising the amino acid sequence ofSEQ ID NO: 168 and/or a light chain variable region (V_(L)CEA)comprising the amino acid sequence of SEQ ID NO: 169.

In a further aspect, the anti-CEA/anti-CD3 bispecific antibody comprisesan Fc domain comprising one or more amino acid substitutions that reducebinding to an Fc receptor and/or effector function. In particular, theanti-CEA/anti-CD3 bispecific antibody comprises an IgG1 Fc domaincomprising the amino acid substitutions L234A, L235A and P329G.

In a particular aspect, the anti-CEA/anti-CD3 bispecific antibodycomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 146, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:147, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 148, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 149.In a further particular embodiment, the bispecific antibody comprises apolypeptide sequence of SEQ ID NO: 146, a polypeptide sequence of SEQ IDNO: 147, a polypeptide sequence of SEQ ID NO: 148 and a polypeptidesequence of SEQ ID NO: 149 (CEA CD3 TCB).

In a further particular aspect, the anti-CEA/anti-CD3 bispecificantibody comprises a polypeptide that is at least 95%, 960%, 97%, 98%,or 99% identical to the sequence of SEQ ID NO: 150, a polypeptide thatis at least 95%, 96%, 976%, 98%, or 99% identical to the sequence of SEQID NO: 151, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 152, and a polypeptide that isat least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 153. In a further particular embodiment, the bispecific antibodycomprises a polypeptide sequence of SEQ ID NO: 150, a polypeptidesequence of SEQ ID NO: 151, a polypeptide sequence of SEQ ID NO: 152 anda polypeptide sequence of SEQ ID NO: 153 (CEACAM5 CD3 TCB).

In another aspect, a pharmaceutical composition comprising a bispecificantibody comprising a first antigen binding domain that specificallybinds to PD1 and a second antigen binding domain that specifically bindsto LAG3 as described herein, and a T-cell activating anti-CD3 bispecificantibody, in particular an anti-CEA/anti-CD3 bispecific antibody isprovided. In a particular aspect, the pharmaceutical composition is foruse in the combined, sequential or simultaneous treatment of a disease,in particular for the treatment of cancer. More particularly, thecomposition is for use in the treatment of solid tumors.

In another aspect, the invention provides a method for treating ordelaying progression of cancer in an individual comprising administeringto the subject an effective amount of bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 as describedherein, in combination with a T-cell activating anti-CD3 bispecificantibody, in particular an anti-CEA/anti-CD3 bispecific antibody oranti-FolR1/anti-CD3 bispecific antibody.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of fusion protein used, the type ofdisorder or treatment, and other factors discussed above. The bispecificantibodies are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the bispecific antibody can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper that ispierceable by a hypodermic injection needle). At least one active agentin the composition is a bispecific antibody comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 as defined herein before.

The label or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises the bispecific antibody of theinvention; and (b) a second container with a composition containedtherein, wherein the composition comprises a further cytotoxic orotherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition.

Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

TABLE C (SEQUENCES) SEQ ID NO: Name Sequence 1heavy chain HVR-H1, PD1-0103 GFSFSSY 2 heavy chain HVR-H2, PD1-0103 GGR3 heavy chain HVR-H3, PD1-0103 TGRVYFALD 4 light chain HVR-L1, PD1-0103SESVDT3DNSF 5 light chain HVR-L2, PD1-0103 RSS 6light chain HVR-L3, PD1-0103 NYDVPW 7 heavy chain variable domain VH,EVILVESGGGLVKPGGSLKLSCAASGFSFSSYTM PD1-0103SWVRQTPEKRLDWVATISGGGRDIYYPDSVKGRF TISRDNAKNTLYLEMSSLMSEDTALYYCVLLTGRVYFALDSWGQGTSVTVSS 8 light chain variable domain VL,KIVLTQSPASLPVSLGQRATISCRASESVDTSDN PD1-0103SFIHWYQQRPGQSPKLLIYRSSTLESGVPARFSG SGSRTDFTLTIDPVEADDVATYYCQQNYDVPWTFGGGTKLEIK 9 humanized variant-heavy chainEVQLLESGGGLVQPGGSLRLSCAASGFSFSSYTM  variable domain VH of PD1-SWVRQAPGKGLEWVATISGGGRDIYYPDSVKGRF  0103_01 (PD1 0376)TISRDNSKNTLYLQMNSLRAEDTAVYYCVLLTGR VYFALDSWGQGTLVTVSS 10humanized variant-light chain DIVMTQSPDSLAVSLGERATINCKASESVDTSDN variable domain VL of PD1- SFIHWYQQKPGQSPKLLIYRSSTLESGVPDRFSG 0103_01 (PD1 0376) SGSGTDFTLTISSLQAEDVAVYYCQQNYDVPWTF GQGTKVEIK 11humanized variant-light chain DVVMTQSPLSLPVTLGQPASISCRASESVDTSDN variable domain VL of PD1- SFIHWYQQRPGQSPRLLIYRSSTLESGVPDRFSG  0103_02SGSGTDFTLKISRVEAEDVGVYYCQQNYDVPWTF GQGTKVEIK 12humanized variant-light chain EIVLTQSPATLSLSPGERATLSCRASESVDTSDN variable domain VL of PD1- SFIHWYQQKPGQSPRLLIYRSSTLESGIPARFSG  0103_03SGSGTDFTLTISSLEPEDFAVYYCQQNYDVPWTF GQGTKVEIK 13humanized variant-light chain EIVLTQSPATLSLSPGERATLSCRASESVDTSDK variable domain VL of PD1- SFIHWYQQKPGQSPRLLIYRSSTLESGIPARFSG  0103_04SGSGTDFTLTISSLEPEDFAVYYCQQNYDVPWTF GQGTKVEIK 14 heavy chain HVR-H1,DYTMN aLAG3(0414) 15 heavy chain HVR-H2, VISWDGGGTY YTDSVKG aLAG3(0414)16 heavy chain HVR-H3, GLTDTTLYGS DY aLAG3(0414) 17light chain HVR-L1, aLAG3(0414) RASQSISSYL N 18light chain HVR-L2, aLAG3(0414) AA STLQS 19light chain HVR-L3, aLAG3(0414) QQTYSSPLT 20heavy chain variable domain VH, EVQLLESGGG LVQPGGSLRL SCAASGFIFDaLAG3(0414) DYTMNWVRQA PGKGLEWVAV ISWDGGGTYYTDSVKGRFTI SRDDFKNTLY LQMNSLRAED TAVYYCAKGL TDTTLYGSDY WGQGTLVTVS S 21light chain variable domain VL, DIQMTQSPSS LSASVGDRVT ITCRASQSISaLAG3(0414) SYLNWYQQKP GKAPKLLIYA ASTLQSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ TYSSPLTFGG GTKVEIK 22heavy chain HVR-H1, aLAG3(0403) DYTMH 23 heavy chain HVR-H2, aLAG3(0403)LVSWDGGGTY YTNSVKG 24 heavy chain HVR-H3, aLAG3(0403) AITDTSLYGY DY 25light chain HVR-L1, aLAG3(0403) RASQSISSYL N 26light chain HVR-L2, aLAG3(0403) AASSLQS 27light chain HVR-L3, aLAG3(0403) QQTYSTPLT 28heavy chain variable domain VH, EVQLLESGGG LVQPGGSLRL SCAASGFTFDaLAG3(0403) DYTMHWVRQA PGKGLEWVSL VSWDGGGTYYTNSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYFCAKAI TDTSLYGYDY WGQGILVTVS S 29light chain variable domain VL, DIQMTQSPSS LSASVGDRVT ITCRASQSISaLAG3(0403) SYLNWYQQKP GNAPXLLIYA ASSLQSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ TYSTPLTFGG GTKVEIK 30heavy chain HVR-H1, aLAG3(0411) DYTMN 31 heavy chain HVR-H2, aLAG3(0411)VISWDGGATY YADSVKG 32 heavy chain HVR-H3, aLAG3(0411) GLTDDTLYGS DY 33light chain HVR-L1, aLAG3(0411) RASQSIVSYL N 34light chain HVR-L2, aLAG3(0411) ASSSLQS 35light chain HVR-L3, aLAG3(0411) QQTYSTPLT 36heavy chain variable domain VH, EVHLLESGGG LVQPGGSLRL SCAASGFIVDaLAG3(0411) DYTMNWVRQA PGKGLEWVSV ISWDGGATYYADSVKGRFTI SRDDFKNTLY LQMNSLRAED TAVYYCAKGL TDDTLYGSDY WGQGTLVTVS S 37light chain variable domain VL, DIQMTQSPSS LSASVGDRVT ITCRASQSIVaLAG3(0411) SYLNWYQQKP GKAPKLLIYA SSSLQSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ TYSTPLTFGG GTKVEIK 38heavy chain HVR-H1, DYAKS aLAG3(0417) 39 heavy chain HVR-H2,GIDNSGYYTY YTDSVKG aLAG3(0417) 40 heavy chain HVR-H3, THSGLIVNDA FDIaLAG3(0417) 41 light chain HVR-L1, aLAG3(0417) RASQSISSYL N  42light chain HVR-L2, aLAG3(0417) AASSLQS 43light chain HVR-L3, aLAG3(041) QQTYSTPLT 44heavy chain variable domain VH, EVQLVESGGG LVQPGGSLRL ACAASGFTFSaLAG3(0417) DYAMSWVRQA PGKGLEWVSG IDNSGYYTYYTDSVKGRFTI SRDDVKNTLY LQMNSLRAED TAVYLCTKTH SGLIVNDAFD IWGQGTMVTV SS 45light chain variable domain VL, DIQMTQSPSS LSASVGDRVT ITCRASQSISaLAG3(0417) SYLNWYQQKP GKAPKLLIYA ASSLQSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ TYSTPLTFGG GTKVEIK 46heavy chain HVR-H1, aLAG3(0416) DYAMS 47 heavy chain HVR-H2, aLAG3(0416)GIDNSGYYTY YTDSVKG 48 heavy chain HVR-H3, aLAG3(04l6) THSGLIVNDA FDI 49light chain HVR-L1, aLAG3(0416) RASQSISSYL N 50light chain HVR-L2, aLAG3(0416) DASSLES 51light chain HVR-L3, aLAG3(0416) QQSYSTPLT 52heavy chain variable domain VH, EVQLVESGGG LVQPGGSLRL ACAASGFTFSaLAG3(0416) DYAMSWVRQA PGKGLEWVSG IDNSGYYTYYTDSVKGRFTI SRDDVKNTLY LQMNSLRAED TAVYLCTKTH SGLIVNDAFD IWGQGTMVTV SS 53light chain variable domain VL, DIQLTQSPSS LSASVGDRVT ITCRASQSISaLAG3(0416) SYLNWYQQKP GKAPKLLIYD ASSLESGVPSRFSGSGSGTD ATLTISSLQP EDFATYYCQQ SYSTPLTFGG GTKVEIK 54heavy chain variable domain VH, QVQLQQWGAG LLKPSETLSL TCAVYGGSFSBMS-986016 (WO2014/008218 DYYWNWIRQP PGKGLEWIGE INHRGSTNSNand US2016/0326248) PSLKSRVTLS LDTSKNQFSL KLRSVTAADTAVYYCAFGYS DYEYNWFDPW GQGTLVTVSS 55 light chain variable domain VLEIVLTQSPAT LSLSPGERAT LSCRASQSIS BMS-986016 (WO2014/008218SYLAWYQQKP GQAPRLLIYD ASNRATGIPA and US2016/0326248)RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGQ GTNLEIK 56heavy chain HVR-H1, MDX25F7 (25F7) DYYWN 57heavy chain HVR-H2, MDX25F7 (25F7) EINHNGNTNSNPSLKS 58heavy chain HVR-H3, MDX25F7 (25F7) GYSDYEYNWF 59light chain HVR-L1, MDX25F7 (25F7) RASQSISSYLA 60light chain HVR-L2, MDX25F7 (25F7) DASNRAT 61light chain HVR-L3, MDX25F7 (25F7) QQRSNWPLT 62heavy chain variable domain VH, QVQLQQWGAG LLKPSETLSL TCAVYGGSFSMDX25F7 (25F7) DYYWNWIRQP PGKGLEWIGE INHNGNTNSN(US2011/0150892 and WO2014/008218) PSLKSRVTLS LDTSKNQFSL KLRSVTAADTAVYYCAFGYS DYEYNWFDPW GQGTLVTVSS 63 light chain variable domain VL,EIVLTQSPAT SYLAWYQQKP RFSGSGSGTD MDX25F7 (25F7)RSNWPLTFGQ LSLSPGERAT GQAPRLLIYD (US2011/0150892 and WO2014/008218)FTLTISSLEP GTNLEIK LSCRASQSIS ASNRATGIPA EDFAVYYCQQ 64heavy chain variable domain VH, QIQLVQSGPE LKKPGETVKI SCKASGFTLThumanized BAP050 NYGMNWVRQT PGKGLKWMGW INTDTGEPTY(LAG525) (US2015/0259420) ADDFKGRFAF SLETSASTAS LQINNLKNASTATYFCARNP PYYYGTNNAE AMDYWGQGTT VTVSS 65light chain variable domain VL, DIQMTQTTSS LSASLGDRVT ISCSSSQDIShumanized BAP050 (LAG525) NYLMWYQQKP DGTVKVLIYY TSTLHLGVPS(US2015/0259420) RFSGSGSGTD YSLTISNLEL EDIATYYCQQ YYNLPWTFGQ GTKVEIK 66heavy chain variable domain VH, QVQLVESGGG VVQPGRSLRL SCAASGFTFSMDX26H10 (26H10) SYGMHWVRQA PGKGLEWVAV IWYDGSNKYY (US 2011/0150892)ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAREW AVASWDYGMD VWGQGTTVTV SS 67light chain variable domain VL, EIVLTQSPGT LSLSPGERAT LSCRASQSVSMDX26H10 (26H10) SSYLAWYQQK PGQAPRLLIY GASSRATGIP (US 2011/0150892)DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPFTFG PGTKVDIK 68human kappa light chain RTVAAPSVFI FPPSDEQLKS GTASVVCLLN constant regionNFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTGQGKSSOVTJ SFNRGEC 69 human lambda light chainQPKAAPSVTL FPPSSEELQA NKATLVCLIS constant regionDFYPGAVTVA WKADSSPVKA GVETTTPSKQ SNNKYAASSY LSLTPEQWKS HRSYSCQVTHEGSTVEKTVA PTECS 70 human heavy chain constantASTKGPSVFP LAPSSKSTSG GTAALGCLVK region derived from IgG1DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 71human heavy chain constant region ASTKGPSVFP LAPSSKSTSG GTAALGCLVKderived from IgG1 with mutations DYFPEPVTVS WNSGALTSGV HTFPAVLQSSL234A, L235A and P329G GLYSLSSVVT VPSSSLGTQT YICNVNEKPSNTKVDNKVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVIT VLHQDWLNGK EYKCKVSNKALGAPIFKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHIT QKSLSLSPG 72human heavy chain constant region ASTKGPSVFP LAPCSRSTSE STAALGCLVKderived from IgG4 DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPSCP APEFLGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVOFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH ODWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTKNQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVESCSVMHE ALHNHYTQKS LSLSLG 73 exemplary human LAG-3 sequenceVPVVWAQEGA PAQLPCSPTI PLQDLSLLRR (without signal sequence)AGVTWQHQPD SGPPAAAPGH PLAPGPHPAA PSSWGPRPRR YTVLSVGPGG LRSGRLPLQPRVQLDERGRQ RGDFSLWLRP ARRADAGEYR AAVHLRDRAI SCRIRLRLGQ ASMTASPPGSLPASDWVILN CSFSRPDRPA SVHWFPNRGQ GPVPVRESPH HHLAESFLFL PQVSPMDSGPWGCILTYRDG FNVSIMYNLT VLGLEPPTPL TVYAGAGSRV GLPCRLPAGV GTRSFLTAKWTPPGGGPDLL VTGDNGDFTL RLEDVSQAQA GTYTCHIHLQ EQQLNATVTL AIITVTPNSFGSPGSLGKLL CFVTPVSGQF RFVWSSLDTP SQRSFSGPWL EAQEAQLLSQ PWOCQLYQGERILGAAVYFT ELSSPGAORS GRAPGALPAG HLLLFLILGV LSLLLLVTGA FGFHLWRRQWRPRRFSALEQ GIHPPQAOSK IEELEQEPEP EPEPEPEPEP EPEPEQL 74human LAG3 Extracellular Domain VPVVWAQEGA PAQLPCSPTI PLQDLSLLRR (ECD)AGVTWQHQPD SGPPAAAPGH PLAPGPHPAA PSSWGPRPRR YTVLSVGPGG LRSGRLPLQPRVQLDERGRQ RGDFSLWLRP ARRADAGEYR AAVHLRDRAI SCRIRLRLGQ ASMTASPPGSLPASDWVILN CSFSRPDRPA SVHWFPNRGQ GPVPVRESPH HHLAESFLFL PQVSPMDSGPWGCILTYRDG FNVSIMYNLT VLGLEPPTPL TVYAGAGSRV GLPCRLPAGV GTRSFLTAKWTPPGGGPDLL VTGDNGDFTL RLEDVSQAQA GTYTCHIHLQ EQQLNATVTL AIITVTPNSFGSPGSLGNLL CEVTPVSGQE RFVWSSLDTP SQRSFSGPWL EAQEAOLLSO PWQCQLYOGERLLGAAVYFT ELSSPGAQRS GRAPGALPAG HL 75 KIEELE (part of LAG3 + KIEELEintracellular domain 76 primer rbHC.up aagcttgcca ccatggagac tgggctgcgctggcttc 77 primer rb-HCf.do ccattggtga gggtqcccga g 78primer BcPCR_FHLC_leader.fw atggacatga gggtccccgc 79primer BcPCR_huCkappa.rev gatttcaact gctcatcaga tggc 80heavy chain HVR-H1, PD1-0098 GYSITSDY 81 heavy chain HVR-H2, PD1-0098YSG 82 heavy chain HVR-H3, PD1-0098 HGSAPWYFD 83light chain HVR-L1, PD1-0098 SQNIVHSDGNTY 84light chain HVR-L2, PD1-0098 KVS 85 light chain HVR-L3, PD1-0098 GSHFPL86 heavy chain variable domain VH, DVQLQESGPGLVKPSQSLSLTCTVT GYSITSDY APD1-0098 WNWIRQFPGDKLEWLGYIT YSG FTNYNPSLKSRISISRDTSKNQFFLQLNSVATEDTATYYCARW HGS APWYFD YWGRGTTLTVSS 87light chain variable domain VL, DVLMTQTPLSLPVSLGDQASISCRS SQNIVHSDGPD1-0098 NTY LEWYLQKPGQSPNLLIY KVS RRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQ GSHFPL T FGAGTKLELK 88heavy chain HVR-H2, PD1-0069 GYTFTDY 89 heavy chain HVR-H2, PD1-0069 YSG90 heavy chain HVR-H3, PD1-0069 GITTGFA 91 light chain HVR-L1, PD1-0069SKGVSTSSYSF 92 light chain HVR-L2, PD1-0069 YAS 93light chain HVR-L3, PD1-0069 SREFPW 94 heavy chain variable domain VH,QVQLQQSGPELVRPGVSVKISCKGS GYTFTDY AM PD1-0069 HWVKQSHARTLEWIGVIST YSGDTNYNQKFKDKA TMTVDKSSSTAYLELARMTSEDSAIYYCARL GIT TGFA YWGQGTLVTVSA 95light chain variable domain VL, DIVLTQSPASLAVSLGQRATISCRA SKGVSTSSYPD1-0069 SF MHWYQQKPRQPPKLLIK YAS YLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCHH SREFPW TF GGGTKLEIK 96 heavy chain1 of 1 +1 PD1/LAGS DIVMTQSPDSLAVSLGERATINCKASESVDTSDN 0799SFIHWYQQKPGQSPKLLIYRSSTLESGVPDRFSG based on PD1(0376/aLAG3(0416)SGSGTDFTLTISSLQAEDVAVYYCQQNYDVPWTF GQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 97 havy chain 2 of 1 + 1 PD1/LAG3EVQLVESGGGLVQPGGSLRLACAASGFTFSDYAM 0799SWVRQAPGKGLEWVSGIDNSGYYTYYTDSVKGRF TISRDDVKNTLYLQMNSLRAEDTAVYLCTKTHSGLIVNDAFDIWGQGTMVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLVSKLTVDKSRWQQGMVFSCSVMHEALHNHYTQKSLSLSPGK 98 light chain 1 of 1 + 1 PD1/LAG3EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYTM 0799SWVRQAPGKGLEWVATISGGGRDIYYPDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVLLTGRVYFALDSWGQGTLVTVSSASVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTKQGLSSPVTKSFNRGEC 99light chain 2 of 1 + 1 PD1/LAG3 DIQLTQSPSSLSASVGDRVTITCRASQSISSYLN 0799WYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSG TDATLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 100 heavy chain 2 of 1 +1 PD1/LAG3 EVQLLESGGGLVQPGGSLRLSCAASGFIFDDYTM 0927NWVRQAPGKGLEWVAVISWDGGGTYYTDSVKGRF based on PD1(0376)/aLAG3(0414)TISRDDFKNTLYLQMNSLRAEDTAVYYCAKGLTD TTLYGSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSKTKVDEKVEPKSCDKTHTCPPCPAPEA AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 101 light chain 2 of 1 +1 PD1/LAG3 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN 0927WYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQTYSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 102 heavy chain 1 of 1 +1 PD1/LAG3  DIVLTQSPASLAVSLGQRATISCRA SKGVSTSSY 0222 SFMHWYQQKPRQPPKLLIK YAS YLESGVPARFSG base on PD1(0069)/SGSGTDFTLNIHPVEEEDAATYYCHH SR E FFW TF aLAG3(MDX25F7)GGGTKLEIKSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K103 heavy chain 2 of 1 + 1 PD1/LAG3 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYW0222 NWIRQPPGKGLEWIGEINHNGNTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDY EYNWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 104QVQLQQSGPELVRPGVSVKISCKGS GYTFTDY AM HWVKQSHARTLEWIGVIST YSGDTNYNQKFKDKA TMTVDKSSSTAYLELARMTSEDSAIYYCARL GIT TGFAYWGQGTLVTVSAASVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC 105light chain 2 of 1 + 1 PD1/LAG3 EIVLTQSPATLSLSPGERATLSCRASQSISSYLA 0222WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 106 heavy chain 1 of 1 +1 PD1/LAG3 DVLMTQTPLSLPVSLCOQASISCRS SQNIVHSDG 0224 NTYLEWYLQKPGQSPNLLIY KVS RRFSGVPDRFS based on PD1(0098)/GSGSGTDFTLKISRVEAEDLGVYYCFQ GSHFPL T aLAG3(MDX25F7)FGAGTKLELKSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQP REPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK107 light chain 1 of 1 + 1 PD1/LAG3 DVQLQESGPGLVKPSQSLSLTCTVT GYSITSDY A0224 WNWIRQFPGDKLEWLGYIT YSG FTNYNPSLKSRISISRDTSKNQFFLQLNSVATEDTATYYCARM HGS APWYFD YWGRGTTLTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 108 aLAG3(0156) heavy chainQVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYW (MDX25F7)NWIRQPPGKGLEWIGEINHNGNTNSNPSLKSRVT LSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSGQPKAPSVFPLAPCCG DTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAH PATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMTSRTPEVTCVVVDVSQDDPEVQFT WYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPL EPKVYTMGPPREELSSRSVSLTCMINGFYPSDIS

The following numbered paragraphs (paras) describe aspects of thepresent invention:

-   1. A bispecific antibody comprising a first antigen binding domain    that specifically binds to programmed cell death protein 1 (PD1) and    a second antigen binding domain that specifically binds to    Lymphocyte activation gene-3 (LAG3), wherein    -   said first antigen binding domain specifically binding to PD1        comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:            1,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:2, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:3; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:4;        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:5, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:6.-   2. The bispecific antibody of para 1, wherein the bispecific    antibody comprises a Fc domain that is an IgG, particularly an IgG1    Fc domain or an IgG4 Fc domain and wherein the Fc domain comprises    one or more amino acid substitution that reduces binding to an Fc    receptor, in particular towards Fcγ receptor.-   3. The bispecific antibody of paras 1 or 2, wherein the second    antigen binding domain that specifically binds to LAG3 comprises    -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:            14.        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:            15, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:            16; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:            17,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:            18, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO: 19; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:22,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:23, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:24; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:25,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:26, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:27; or    -   (c) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:30,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:31, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:32; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:33,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:34, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:35; or    -   (d) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:38,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:39, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:40; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:41,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:42, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:43; or    -   (e) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:46,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:47, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:48; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:49,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:50, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:51.-   4. The bispecific antibody according to any one of paras 1 to 3,    wherein the first antigen-binding domain specifically binding to PD1    comprises    -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        7 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 8, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 10, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 11, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 12, or    -   (e) a VH domain comprising the amino acid sequence of SEQ ID NO:        9 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 13.-   5. The bispecific antibody according to any one of paras 1 to 4,    wherein the second antigen-binding domain specifically binding to    LAG3 comprises    -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        20 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 21, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        28 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 29, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        36 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 37, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        44 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 45, or    -   (e) a VH domain comprising the amino acid sequence of SEQ ID NO:        52 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 53.-   6. The bispecific antibody according to any one of paras 1 to 4,    wherein the second antigen-binding domain specifically binding to    LAG3 comprises    -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        54 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 55, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        62 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 63, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        64 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 65, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        66 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 67.-   7. The bispecific antibody according any one of paras 1 to 5,    wherein    -   the first antigen binding domain specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 9 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 10.    -   and the second antigen binding domain specifically binding to        LAG3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 20 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 21 or a VH domain comprising the amino acid        sequence of SEQ ID NO: 52 and a VL domain comprising the amino        acid sequence of SEQ ID NO: 53.-   8. The bispecific antibody according any one of paras 1 to 5,    wherein    -   the first antigen binding domain specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 9 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 10.    -   and the second antigen binding domain specifically binding to        LAG3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 20 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 21.-   9. The bispecific antibody according any one of paras 1 to 4 or 6,    wherein    -   the first antigen binding domain specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 9 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 10,    -   and the second antigen binding domain specifically binding to        LAG3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 56 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 57.-   10. The bispecific antibody according to any one of paras 1 to 5,    wherein the bispecific antibody is a humanized or chimeric antibody.-   11. The bispecific antibody of any one of paras 1 to 10, wherein the    bispecific antibody comprises an Fc domain of human IgG1 subclass    with the amino acid mutations L234A, L235A and P329G (numbering    according to Kabat EU index).-   12. The bispecific antibody of any one of paras 1 to 11, wherein the    bispecific antibody comprises an Fc domain comprising a modification    promoting the association of the first and second subunit of the Fc    domain.-   13. The bispecific antibody of any one of paras 1 to 12, wherein the    first subunit of the Fc domain comprises knobs and the second    subunit of the Fc domain comprises holes according to the knobs into    holes method.-   14. The bispecific antibody of any one of paras 1 to 13, wherein the    first subunit of the Fc domain comprises the amino acid    substitutions S354C and T366W (EU numbering) and the second subunit    of the Fc domain comprises the amino acid substitutions Y349C, T366S    and Y407V (numbering according to Kabat EU index).-   15. The bispecific antibody of any one of paras 1 to 14, wherein the    bispecific antibody comprises an Fc domain, a first Fab fragment    comprising the antigen binding domain that specifically binds to PD1    and a second Fab fragment comprising the antigen binding domain that    specifically binds to LAG3.-   16. The bispecific antibody of any one of paras 1 to 15, wherein in    one of the Fab fragments the the variable domains VL and VH are    replaced by each other so that the VH domain is part of the light    chain and the VL domain is part of the heavy chain.-   17. The bispecific antibody of paras 15 or 16, wherein in the first    Fab fragment comprising the antigen binding domain that specifically    binds to PD1 the variable domains VL and VH are replaced by each    other.-   18. The bispecific antibody of any one of paras 1 to 17, wherein the    bispecific antibody comprises a Fab fragment wherein in the constant    domain CL the amino acid at position 124 is substituted    independently by lysine (K), arginine (R) or histidine (H)    (numbering according to Kabat EU Index), and in the constant domain    CH1 the amino acids at positions 147 and 213 are substituted    independently by glutamic acid (E) or aspartic acid (D) (numbering    according to Kabat EU index).-   19. The bispecific antibody of any one of paras 15 to 18, wherein in    the second Fab fragment comprising the antigen binding domain that    specifically binds to LAG3 the constant domain CL the amino acid at    position 124 is substituted independently by lysine (K),    arginine (R) or histidine (H) (numbering according to Kabat EU    Index), and in the constant domain CH1 the amino acids at positions    147 and 213 are substituted independently by glutamic acid (E) or    aspartic acid (D) (numbering according to Kabat EU index).-   20. The bispecific antibody of any one of paras 1 to 19, comprising-   (a) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 96, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 98,    -   a second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 97,        and a second light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:99,        or-   (b) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 96, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 98,    -   a second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 100,        and a second light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        101, or-   (c) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 102, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 104,    -   a second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 103,        and a second light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        105, or-   (d) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 106, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 107,    -   a second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 103,        and a second light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        105.-   21. The bispecific antibody of any one of paras 1 to 19, wherein the    bispecific antibody comprises a third Fab fragment comprising an    antigen binding domain that specifically binds to LAG3.-   22. The bispecific antibody of any one of paras 1 to 19 or 21,    wherein the two Fab fragments comprising each an antigen binding    domain that specifically binds to LAG3 are identical.-   23. The bispecific antibody of any one of paras 1 to 19 or 21 or 22,    wherein the Fab fragment comprising the antigen binding domain that    specifically binds to PD1 is fused via a peptide linker to the    C-terminus of one of the heavy chains.-   24. The bispecific antibody of any one of paras 1 to 19 or 21 to 23,    comprising-   (a) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 118, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 115,    -   a second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 119,        and a second light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        101, or-   (b) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 120, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 115,    -   a second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 121,        and a second light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:99,        or-   (c) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 122, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 115,    -   a second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 103,        and a second light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:        105.-   25. The bispecific antibody of any one of paras 1 to 19 or 21 to 23,    wherein the bispecific antibody comprises a fourth Fab fragment    comprising an antigen binding domain that specifically binds to PD1.-   26. The bispecific antibody of any one of paras 1 to 19 or 21 to 23    or 25, wherein the two Fab fragments comprising each an antigen    binding domain that specifically binds to PD1 are identical.-   27. The bispecific antibody of any one of paras 1 to 19 or 21 to 23    or 25 or 26, wherein the two Fab fragments comprising each an    antigen binding domain that specifically binds to PD1 are each fused    via a peptide linker to the C-terminus to one of the heavy chains,    respectively.-   28. The bispecific antibody of any one of paras 1 to 19 or 21 to 23    or 25 to 27, comprising-   (a) two heavy chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 114, two    first light chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 115, and    two second light chains comprising each an amino acid sequence with    at least 95% sequence identity to the sequence of SEQ ID NO: 101, or-   (b) two heavy chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 116, two    first light chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 115, and    two second light chains comprising each an amino acid sequence with    at least 95% sequence identity to the sequence of SEQ ID NO:99, or-   (c) two heavy chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 117, two    first light chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 115, and    two second light chains comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 105.-   29. The bispecific antibody of any one of paras 1 to 14, wherein the    bispecific antibody comprises an Fc domain, two Fab fragments    comprising each an antigen binding domain that specifically binds to    LAG3 and a single chain Fab (scFab) comprising the antigen binding    domain that specifically binds to PD1.-   30. The bispecific antibody of any one of paras 1 to 14 or 29,    wherein the scFab comprising an antigen binding domain that    specifically binds to PD1 is fused via a peptide linker to the    C-terminus to one of the heavy chains.-   31. The bispecific antibody of any one of paras 1 to 14 or 29 or 30,    comprising-   (a) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 123, a    second heavy chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 119, and two    light chains comprising each an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 101, or-   (b) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 124, a    second heavy chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 121, and two    light chains comprising each an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO:99, or-   (c) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 125, a    second heavy chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 103, and a    second light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 105.-   32. The bispecific antibody of any one of paras 1 to 14, wherein the    bispecific antibody comprises an Fc domain, two Fab fragments    comprising each an antigen binding domain that specifically binds to    LAG3 and a VH and VL domain comprising the antigen binding domain    that specifically binds to PD1.-   33. The bispecific antibody of any one of paras 1 to 14 or 32,    wherein the VH domain of the antigen binding domain that    specifically binds to PD1 is fused via a peptide linker to the    C-terminus of one of the heavy chains and the VL domain of the    antigen binding domain that specifically binds to PD1 is fused via a    peptide linker to the C-terminus of the other one of the heavy    chains.-   34. The bispecific antibody of any one of paras 1 to 14 or 32 or 33,    comprising a first heavy chain comprising an amino acid sequence    with at least 95% sequence identity to the sequence of SEQ ID NO:    126, a second heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 127, and    two light chains comprising each an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 109.-   35. A polynucleotide encoding the bispecific antibody of any one of    paras 1 to 34.-   36. A vector, particularly an expression vector, comprising the    polynucleotide according to para 35.-   37. A prokaryotic or eukaryotic host cell comprising the    polynucleotide according to para 35 or the vector according to para    36.-   38. A method of producing the bispecific antibody according to paras    1 to 34, comprising culturing the host cell of para 37 under    conditions suitable for the expression of the bispecific antibody    and recovering the bispecific antibody from the culture.-   39. A pharmaceutical composition comprising the bispecific antibody    according to any one of paras 1 to 34 and at least one    pharmaceutically acceptable excipient.-   40. The bispecific antibody according to any one of paras 1 to 34 or    the pharmaceutical composition according to para 39 for use as a    medicament.-   41. The bispecific antibody according to any one of paras 1 to 34 or    the pharmaceutical composition according to para 39 for use    -   i) in the modulation of immune responses, such as restoring T        cell activity,    -   ii) in stimulating a T cell response,    -   iii) in the treatment of infections,    -   iv) in the treatment of cancer.    -   v) in delaying progression of cancer,    -   vi) in prolonging the survival of a patient suffering from        cancer.-   42. The bispecific antibody according to any one of paras 1 to 34 or    the pharmaceutical composition according to para 39 for use in the    prevention or treatment of cancer.-   43. The bispecific antibody according to any one of paras 1 to 34 or    the pharmaceutical composition according to para 39 for use in the    treatment of a chronic viral infection.-   44. The bispecific antibody according to any one of paras 1 to 34 or    the pharmaceutical composition according to para 39 for use in the    prevention or treatment of cancer, wherein the bispecific antibody    is administered in combination with a chemotherapeutic agent,    radiation and/or other agents for use in cancer immunotherapy.-   45. A method of inhibiting the growth of tumor cells in an    individual comprising administering to the individual an effective    amount of the bispecific antibody according to any one of claims 1    to 34 to inhibit the growth of the tumor cells.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Amino acids of antibody chains are numbered and referred to according tothe numbering systems according to Kabat (Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) as definedabove.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular Cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The 600-1800 bp long gene segments, which wereflanked by singular restriction endonuclease cleavage sites, wereassembled by annealing and ligating oligonucleotides including PCRamplification and subsequently cloned via the indicated restrictionsites e.g. KpnI/SacI or AscI/PacI into a pPCRScript (Stratagene) basedpGA4 cloning vector. The DNA sequences of the subcloned gene fragmentswere confirmed by DNA sequencing. Gene synthesis fragments were orderedaccording to given specifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described antibodies, variants of expressionplasmids for transient expression (e.g. in HEK293) cells based either ona cDNA organization with or without a CMV-Intron A promoter or on agenomic organization with a CMV promoter were applied.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this        plasmid in E. coli, and    -   a ß-lactamase gene which confers ampicillin resistance in E.        coli.

The transcription unit of the antibody gene was composed of thefollowing elements:

-   -   unique restriction site(s) at the 5′ end    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   followed by the Intron A sequence in the case of the cDNA        organization,    -   a 5′-untranslated region of a human antibody gene,    -   an immunoglobulin heavy chain signal sequence,    -   the human antibody chain (wildtype or with domain exchange)        either as cDNA or as genomic organization with the        immunoglobulin exon-intron organization    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

The fusion genes comprising the antibody chains as described below weregenerated by PCR and/or gene synthesis and assembled by knownrecombinant methods and techniques by connection of the accordingnucleic acid segments e.g. using unique restriction sites in therespective vectors. The subcloned nucleic acid sequences were verifiedby DNA sequencing. For transient transfections larger quantities of theplasmids were prepared by plasmid preparation from transformed E. colicultures (Nucleobond AX, Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.). John Wiley &Sons, Inc.

Multispecific antibodies were expressed by transient co-transfection ofthe respective expression plasmids in adherently growing HEK293-EBNA orin HEK29-F cells growing in suspension as described below.

Transient Transfections in HEK293 System

All antibodies and bispecific antibodies were generated by transienttransfection of 293F cells using the Freestyle system (ThermoFisher).Here the 293F cells were cultivated in F17 Medium, transfected with293Free (Novagene) and feeded after 4 hours with VPA 4 mM and Feed 7 and0.6% Glucose after 16 h. Further the Expi293F™ Expression System Kit(ThermoFisher) was used. Here the Expi293F™ cells were cultivated inExpi293™ Expression Medium and transfected using ExpiFectamine™ 293Transfection Kit according manufactuer's instructions. Due to theimproved stability and purity and reduced aggregation tendency of theCrossMAb^(Vh-VL) bispecific antibodies with additionally introducedcharged pairs of amino acids in the CH1/CL interface (see positions inthe respective sequences for further detail) no adjustments of plasmidratio have been employed. Therefore the relative plasmid ratio of1:1:1:1 for 1+1 CrossMab or 1:1:1 for 2+2 CrossMab was used for theco-transfection of LC, HC, crossed LC and crossed HC plasmids. Cellsupernatants were harvested after 7 days and purified by standardmethods.

Protein Determination

The protein concentration of purified antibodies and derivatives wasdetermined by determining the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of antibodies and derivatives in cell culturesupernatants was estimated by immunoprecipitation with Protein AAgarose-beads (Roche). 60 μL Protein A Agarose beads were washed threetimes in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40).Subsequently, 1-15 mL cell culture supernatant was applied to theProtein A Agarose beads pre-equilibrated in TBS-NP40. After incubationfor at 1 hour at room temperature the beads were washed on anUltrafree-MC-filter column (Amicon) once with 0.5 mL TBS-NP40, twicewith 0.5 mL 2× phosphate buffered saline (2×PBS, Roche) and briefly fourtimes with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody was eluted byaddition of 35 μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of thesample was combined with NuPAGE® Sample Reducing Agent or leftunreduced, respectively, and heated for 10 min at 70° C. Consequently,5-30 μl were applied to a 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen)(with MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE®Antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE)and stained with Coomassie Blue.

The concentration of antibodies and derivatives in cell culturesupernatants was quantitatively measured by affinity HPLCchromatography. Briefly, cell culture supernatants containing antibodiesand derivatives that bind to Protein A were applied to an AppliedBiosystems Poros A/20 column in 200 mM KH2PO4, 100 mM sodium citrate, pH7.4 and eluted from the matrix with 200 mM NaCl, 100 mM citric acid, pH2.5 on an Agilent HPLC 1100 system. The eluted protein was quantified byUV absorbance and integration of peak areas. A purified standard IgG1antibody served as a standard.

Alternatively, the concentration of antibodies and derivatives in cellculture supernatants was measured by Sandwich-IgG-ELISA. Briefly,StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche)are coated with 100 μL/well biotinylated anti-human IgG capture moleculeF(ab′)2<h-Fcγ> BI (Dianova) at 0.1 μg/mL for 1 hour at room temperatureor alternatively overnight at 4° C. and subsequently washed three timeswith 200 μL/well PBS, 0.05% Tween (PBST, Sigma). 100 μL/well of adilution series in PBS (Sigma) of the respective antibody containingcell culture supernatants was added to the wells and incubated for 1-2hour on a microtiter plate shaker at room temperature. The wells werewashed three times with 200 μL/well PBST and bound antibody was detectedwith 100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as the detectionantibody for 1-2 hours on a microtiter plate shaker at room temperature.Unbound detection antibody was washed away three times with 200 μL/wellPBST and the bound detection antibody was detected by addition of 100 μLABTS/well. Determination of absorbance was performed on a Tecan FluorSpectrometer at a measurement wavelength of 405 nm (reference wavelength492 nm).

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE healthcare) and washed with PBS. Elution ofantibodies was achieved at pH 2.8 followed by immediate neutralizationof the sample. Aggregated protein was separated from monomericantibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomericantibody fractions were pooled, concentrated (if required) using e.g., aMILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen andstored at −20° C. or −80° C. Part of the samples were provided forsubsequent protein analytics and analytical characterization e.g. bySDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer was used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed by HPLCchromatography. Briefly, Protein A purified antibodies were applied to aTosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5on an Agilent HPLC 1100 system or to a Superdex 200 column (GEHealthcare) in 2×PBS on a Dionex HPLC-System. The eluted protein wasquantified by UV absorbance and integration of peak areas. BioRad GelFiltration Standard 151-1901 served as a standard.

Mass Spectrometry

This section describes the characterization of the multispecificantibodies with VH/VL exchange (VH/VL CrossMabs) with emphasis on theircorrect assembly. The expected primary structures were analyzed byelectrospray ionization mass spectrometry (ESI-MS) of the deglycosylatedintact CrossMabs and deglycosylated/plasmin digested or alternativelydeglycosylated/limited LysC digested CrossMabs.

The VH/VL CrossMabs were deglycosylated with N-Glycosidase F in aphosphate or Tris buffer at 37° C. for up to 17 h at a proteinconcentration of 1 mg/ml. The plasmin or limited LysC (Roche) digestionswere performed with 100 pig deglycosylated VH/VL CrossMabs in a Trisbuffer pH 8 at room temperature for 120 hours and at 37° C. for 40 min,respectively. Prior to mass spectrometry the samples were desalted viaHPLC on a Sephadex G25 column (GE Healthcare). The total mass wasdetermined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik)equipped with a TriVersa NanoMate source (Advion).

Determination of Binding and Binding Affinity of MultispecificAntibodies to the Respective Antigens Using Surface Plasmon Resonance(SPR) (BIACORE)

Binding of the generated antibodies to the respective antigens isinvestigated by surface plasmon resonance using a BIACORE instrument (GEHealthcare Biosciences AB, Uppsala, Sweden). The respective BiacoreEvaluation Software is used for analysis of sensorgrams and forcalculation of affinity data.

Example 1 Generation of Anti-PD-1 Antibodies Immunization of Mice

NMRI mice were immunized genetically, using a plasmid expression vectorcoding for full-length human PD-1 by intradermal application of 100 ugvector DNA (plasmid15300_hPD1-fl), followed by Electroporation (2 squarepulses of 1000 V/cm, duration 0.1 ms, interval 0.125 s; followed by 4square pulses of 287.5 V/cm, duration 10 ms, interval 0.125 s. Micereceived either 6 consecutive immunizations at days 0, 14, 28, 42, 56,70, and 84. Blood was taken at days 36, 78 and 92 and serum prepared,which was used for titer determination by ELISA (see below). Animalswith highest titers were selected for boosting at day 96, by intravenousinjection of 50 ug of recombinant human PD1 human Fc chimera, andmonoclonal antibodies were isolated by hybridoma technology, by fusionof splenocytes to myeloma cell line 3 days after boost.

Determination of Serum Titers (ELISA)

Human recombinant PD1 human Fc chimera was immobilized on a 96-well NUNCMaxisorp plate at 0.3 ug/ml, 100 ul/well, in PBS, followed by: blockingof the plate with 2% Crotein C in PBS, 200 ul/well; application ofserial dilutions of antisera, in duplicates, in 0.5% Crotein C in PBS,100 ul/well; detection with HRP-conjugated goat anti-mouse antibody(Jackson Immunoresearch/Dianova 115-036-071; 1/16 000). For all steps,plates were incubated for 1 h at 37° C. Between all steps, plates werewashed 3 times with 0.05% Tween 20 in PBS. Signal was developed byaddition of BM Blue POD Substrate soluble (Roche), 100 ul/well; andstopped by addition of 1 M HCl, 100 ul/well. Absorbance was read out at450 nm, against 690 nm as reference. Titer was defined as dilution ofantisera resulting in half-maximal signal.

Example 2 Characterization Anti-PD1 Antibodies/Binding of Anti-PD1Antibodies to Human PD1 ELISA for hu PD1

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated PD1-ECD-AviHis and incubated at 4°C. over night. After washing (3×90 μl/well with PBST-buffer) 25 μl antiPD1 samples or reference antibodies (human anti PD1; Roche/mouse antiPD1; Biolegend; cat.:329912) were added and incubated 1 h at RT. Afterwashing (3×90 μl/well with PBST-buffer) 25 μl/well goat-anti-humanH+L-POD (JIR, JIR109-036-088)/Sheep-anti-mouse-POD (GE Healthcare;NA9310) was added in 1:2000/1:1000 dilution and incubated at RT for 1 hon shaker. After washing (3×90 μl/well with PBST-buffer) 25 μl/well TMBsubstrate (Roche Catalogue No. 11835033001) was added and incubateduntil OD 2-3. Measurement took place at 370/492 nm.

ELISA results are listed as EC₅₀-values [ng/ml] in Summary Tables 1 and2 below.

Cell ELISA for PD1

Adherent CHO-K cell line stably transfected with plasmid15311_hPD1-fl_pUC_Neo coding for full-length human PD1 and selectionwith G418 (Neomycin resistance marker on plasmid) were seeded at aconcentration of 0.01×10E6 cells/well in 384-well flat bottom plates andgrown over night.

The next day 25 μl/well PD1 sample or human anti PD1 (Roche)/mouse antiPD1 (Biolegend; cat.:329912) reference antibody were added and incubatedfor 2 h at 4° C. (to avoid internalization). After washing carefully(1×90 μl/well PBST) cells were fixed by adding 30 μl/well 0.05%Glutaraldehyde (Sigma, Cat. No: G5882, 25%) diluted in 1×PBS-buffer andincubated for 10 min at RT. After washing (3×90 μl/well PBST) 25 μl/wellsecondary antibody was added for detection: goat-anti-human H+L-POD(JIR, JIR109-036-088)/Sheep-anti-mouse-POD (GE NA9310) followed by 1 hincubation at RT on shaker. After washing (3×90 μl/well PBST) 25 μl/wellTMB substrate solution (Roche 11835033001) was added and incubated untilOD 1.0-2.0. Plates were measured at 370/492 nm.

Cell ELISA results are listed as “EC₅₀ CHO-PD1”-values [ng/ml] in Table2 below.

ELISA for Cyno PD1

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated cynoPD1-ECD-Biotin and incubated at4° C. over night. After washing (3×90 μl/well with PBST-buffer) 25 μlanti PD1 samples or reference antibodies (human anti PD1; Roche) wereadded and incubated 1 h at RT on shaker. After washing (3×90 μl/wellwith PBST-buffer) 25 μL/well goat-anti-human H+L-POD (JIR,JIR109-036-088) was added in 1:1000 dilution and incubated at RT for 1 hon shaker. After washing (3×90 μl/well with PBST-buffer) 25 μl/well TMBsubstrate (Roche, 11835033001) was added and incubated until OD 2-3.Measurement took place at 370/492 nm.

ELISA results are listed as EC₅₀-values [ng/ml] in Summary Table 1 and 2below.

PD1 Ligand 1 Replacing Assay

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated PD1-ECD-AviHis and incubated at 4°C. over night. After washing (3×90 μl/well with PBST-buffer) 25 μl antiPD1 samples or reference antibodies (mouse anti PD1; Biolegend;cat.:329912) were added and incubated 1 h at RT on shaker. After washing(3×90 μl/well with PBST-buffer) 25 μl/well PD-L (Recombinant humanB7-H1/PD-L1 Fc Chimera; 156-B7, R&D) was added and incubated 1 h at RTon shaker. After washing (3×90 μl/well with PBST-buffer) 25 μl/wellgoat-anti-human H+L-POD (JIR, 109-036-088) was added in 1:1000 dilutionand incubated at RT for 1 h on shaker. After washing (3×90 μl/well withPBST-buffer) 25 μl/well TMB substrate (Roche, 11835033001) was added andincubated until OD 2-3. Measurement took place at 370/492 nm.

ELISA results are listed as IC₅₀-values [ng/ml] in summary Table 1below.

PD Ligand 2 Replacing Assay

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated PD1-ECD-AviHis and incubated at 4°C. over night. After washing (3×90 μl/well with PBST-buffer) 25 μl antiPD1 samples or reference antibodies (mouse anti huPD1; Roche) were addedand incubated 1 h at RT on shaker. After washing (3×90 μl/well withPBST-buffer) 25 μl/well PD-L2 (Recombinant human B7-DC/PD-L2 Fc Chimera;1224-PL-100, R&D) was added and incubated 1 h at RT on shaker. Afterwashing (3×90 μl/well with PBST-buffer) 25 μl/well goat-anti-humanH+L-POD (JIR, 109-036-088) was added in 1:2000 dilution and incubated atRT for 1 h on shaker. After washing (3×90 μl/well with PBST-buffer) 25μl/well TMB (tetramethylbenzidine) substrate (Roche, #11835033001) wasadded and incubated until OD 2-3. Measurement took place at 370/492 nm.

ELISA results are listed as IC₅₀-values [ng/ml] in summary Table 1below.

Epitope Mapping ELISA/Binding Competition Assay

Nunc maxisorp plates (Nunc #464718) were coated with 25 μL/well captureantibody (goat anti mouse IgG; JIR; 115-006-071) and incubated for 1 hat RT on shaker. After washing (3×90 μl/well with PBST-buffer) plateswere blocked for 1 h with 2% BSA containing PBS buffer at RT on shaker.After washing (3×90 μl/well with PBST-buffer) 25 μl mouse anti PD1samples were added and incubated 1 h at RT on shaker. After washing(3×90 μl/well with PBST-buffer) capture antibody was blocked by 30μl/well mouse IgG (JIR; 015-000-003) for 1 h at RT on shaker. At thesame time biotinylated PD1-ECD-AviHis was preincubated with secondsample antibody for 1 h at RT on shaker. After washing assay plate (3×90μl/well with PBST-buffer) the PD1 antibody mix was transferred to assayplate and incubated at RT for 1 h on shaker. After washing (3×90 μl/wellwith PBST-buffer) 25 μl/well streptavidin POD (Roche, #11089153001) wasadded in 1:4000 dilution and incubated at RT for 1 h on shaker. Afterwashing (3×90 μl/well with PBST-buffer) 25 μl/well TMB substrate (Roche,#11089153001) was added and incubated until OD 1.5-2.5. Measurement tookplace at 370/492 nm. Epitope groups were defined by hierarchicalclustering against reference antibodies.

TABLE 1 Binding, PD-L1 inhibition and epitope region groups of exemplaryantibodies (ELISA) ELISA ELISA Epitope ELISA ELISA PD-L1 PD-L2 regionhuPD1 cyPD1 inhibition inhibition group (By EC₅₀ EC₅₀ IC₅₀ IC₅₀competion Antibody [ng/ml] [ng/ml] [ng/ml] [ng/ml] assay) PD1-0050 17.99.8 128 34 1 PD1-0069 45.7 22.7 225 89 6 PD1-0073 15.1 8.3 124 65 5PD1-0078 26.3 22.4 x 86 2 PD1-0098 50.8 54.6 174 45 5 PD1-0102 34.252.7 >35.5 μg/ml 140 4 PD1-0103 33.7 36.9 182 51 5

TABLE 2 Biochemial- and Cell-binding of humanized PD1 antibodies derivedfrom parental mouse antibody PD1-0103 (ELISA) Humanized ELISA huPD1ELISA cyPD1 ELISA CHO-PD1 antibody EC₅₀ [ng/ml] EC₅₀ [ng/ml] EC₅₀[ng/ml] PD1-103- 11 8.3 10.1 0312 PD1-103- 15 11 10.8 0313 PD1-103- 118.3 7.7 0314 PD1-103- 10 7.9 7.3 0315

Biacore Characterization of the Humanized Anti-PD-1 Antibodies

A surface plasmon resonance (SPR) based assay has been used to determinethe kinetic parameters of the binding between several murine PD1 bindersas well as commercial human PD1 binding references. Therefore, ananti-human IgG was immobilized by amine coupling to the surface of a(Biacore) CM5 sensor chip. The samples were then captured and hu PD1-ECDwas bound to them. The sensor chip surface was regenerated after eachanalysis cycle. The equilibrium constant and kinetic rate constants werefinally gained by fitting the data to a 1:1 langmuir interaction model.

About 2000 response units (RU) of 20 μg-ml anti-human IgG (GE Healthcare#BR-1008-39) were coupled onto the flow cells 1 and 2 (alternatively: 3and 4) of a CM5 sensor chip in a Biacore T200 at pH 5.0 by using anamine coupling kit supplied by GE Healthcare.

The sample and running buffer was HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.05% v/v Surfactant P20, pH 7.4). Flow cell temperature wasset to 25° C. and sample compartment temperature to 12° C. The systemwas primed with running buffer.

The samples were injected for 20 seconds with a concentration of 10 nMand bound to the second flow cell. Then a complete set of human PD1-ECDconcentrations (144 nM, 48 nM, 16 nM, 5.33 nM, 1.78 nM, 0.59 nM, 0.20 nMand 0 nM) was injected over each sample for 120 s followed by adissociation time of 30/300 s and two 20 s regeneration steps with 3 MMgCl₂, of which the last one contained an “extra wash after injection”with running buffer.

Finally the double referenced data was fitted to a 1:1 Langmuirinteraction model with the Biacore T200 Evaluation Software. ResultingK_(D), k_(a) and k_(d) values are shown in Table 3.

TABLE 3 Kinetic rate constants and equilibrium constants for chimericPD1-0103 and humanized PD1-Abs determined by Biacore Ligand k_(a)[M⁻¹s⁻¹] k_(d) [s⁻¹] K_(D) [nM] chimeric PD1-0103 3.86E+05 3.07E−04 0.8PD1-0103-0312 1.95E+05 3.45E−04 1.8 PD1-0103-0313 1.60E+05 3.67E−04 2.3PD1-0103-0314 1.87E+05 2.79E−04 1.5 PD1-0103-0315 1.89E+05 2.91E−04 1.5

As shown in Table 3, all the humanized versions of chimeric PD1-0103(generation see Example 6) display kinetic properties similar to theparental antibody (chimeric PD1-0103).

Kinetics

A CM5 sensor series S was mounted into the Biacore 4000 System and thedetection spots were hydrodynamically addressed according to themanufacturer's instructions.

The polyclonal rabbit IgG antibody <IgGFCγM>R (Jackson ImmunoResearchLaboratories Inc.) was immobilized at 10 000 Ru on the detection spots 1and 5 in the flow cells 1, 2, 3 and 4. Coupling was done via EDC/NHSchemistry according to the manufacturer's instructions. The remainingspots in the flow cells served as a reference. The sample buffer was thesystem buffer supplemented with 1 mg/ml carboxymethyldextrane.

In one embodiment the assay was driven at 25° C. In another embodimentthe assay was driven at 37° C. 50 nM of each murine monoclonal antibodywas captured on the sensor surface by a 1 min injection at 10 μl/min.Subsequently the respective antigens were injected in a concentrationseries of 100 nM, 2×33 nM, 11 nM, 4 nM, 1 nM and system buffer 0 nM at30 μl/min for 4 min association phase time. The dissociation wasmonitored for another 4 min. The capture system was regenerated using a3 min injection of 10 mM glycine pH 1.5 at 30 μl/min. The relevantkinetic data was calculated using the Biacore evaluation softwareaccording to the manufacturer's instructions.

Epitope Mapping

A Biacore 4000 instrument was mounted with a Biacore CAP sensor and wasprepared like recommended by the manufacturer. The instrument buffer wasHBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween20). The instrument was running at 25° C.

All samples were diluted in system buffer. A 35 kDa biotinylated antigenPD1-ECD-AviHis was captured at 200 RU on the CAP sensor surface by a 1min injection at 301l/min in the flow cells 1, 2, 3 and 4 in the spots 1and 5. Spots 2, 3 and 4 served as a reference. In another embodiment, a35 kDa biotinylated antigen PD1-ECD-AviHis was captured at 200 RU on theCAP sensor in the same manner.

Subsequently a primary antibody was injected at 100 nM for 3 min at 30μl/min followed by the injection of a secondary antibody at 100 nM for 3min at 30 μl/min. The primary antibody was injected until fullsaturation of the surface presented antigen. At the end of the primaryand secondary antibody injection phases report points “Binding Late”(BL) were set to monitor the binding response of the respectiveantibodies. The Molar Ratio, a quotient between the secondary antibodybinding response “BL2” and the primary antibody response “BL1” wascalculated. The Molar Ratio was used as an indicator of the antigenaccessibility of the secondary antibody, when the antigen was alreadycomplexed by the primary antibody.

The complexes were completely removed from the sensor surface by aninjection for 2 min at 30μ/min 2M guanidine-HCL 250 mM NaOH regenerationbuffer as recommended by the manufacturer, followed by a 1 min injectionat 30 μl/min of system buffer.

Example 3 Effect of Different Anti-PD-1 Antibodies on CytokineProduction in a Mixed Lymphocyte Reaction (MLR)

3A) The Mixed Lymphocyte Reaction (MLR) is a immune cell assay whichmeasures the activation of lymphocytes from one individual (donor X) tolymphocytes from another individual (donor Y). A mixed lymphocytereaction was used to demonstrate the effect of blocking the PD1 pathwayto lymphocyte effector cells. T cells in the assay were tested foractivation and their IFNγ secretion in the presence or absence of ananti-PD1 mAbs.

To perform an allogeneic MLR, peripheral blood mononuclear cells (PBMCs)from at least four healthy donors of unknown HLA type were isolated bydensity gradient centrifugation using Leukosep (Greiner Bio One, 227288). Briefly, heparinized blood samples were diluted with the threefold volume of PBS and 25 ml aliquots of the diluted blood were layeredin 50 ml Leukosep tubes. After centrifugation at 800×g for 15 min atroom temperature (w/o break) the lymphocyte containing fractions wereharvested, washed in PBS and used directly in functional assay orresuspended in freezing medium (10% DMSO, 90% FCS) at 1.0E+07 cells/mland stored in liquid nitrogen. Individual 2-way MLR reactions were setup by mixing PBMCs from two different donors at a 1:1stimulator/responder cell ratio and co-cultures were done at least induplicate in flat-bottomed 96-well plates for 6 days at 37° C., 5% CO₂,in the presence or w/o of a different concentration range of purifiedanti-PD1 monoclonal antibodies PD1-0050, PD1-0069, PD1-0073, PD1-0078,PD1-0098. PD1-0102, PD1-0103. As reference anti-PD1 antibodies,antibodies comprising the VH and VL domains of either nivolumab (alsoknown as MDX-5C4 or MDX-1106) or pembrolizumab (also known as MK-3475 orOrg 1.09A) were synthesized and cloned with backbones of human IgG1(with mutations L234A, L235A and P329G (EU index of Kabat)). Either noantibody or an isotype control antibody was used as a negative controland rec hu IL-2 (20 EU/ml) was used as positive control. After day 6 100μl of medium was taken from each culture for cytokine measurement. Thelevels of IFN-gamma were measured using OptEIA ELISA kit (BDBiosciences).

The results are shown in Table 4 (IFNγ secretion/release). The anti-PD1monoclonal antibodies promoted T cell activation and IFNγ secretion inconcentration dependent manner. The value of % increase of IFNγsecretion was calculated in relation to IFNγ production of MLR w/oadding of any blocking mAbs (basal allogeneic stimulation induced IFNγvalue as E−c) and MLR with adding of 20 EU/ml rec hu IL-2 (positivecontrol=100% IFNγ value as E+c) and was calculated according to formula:Rel.Stimulation [%]=((Exampke−E−c)/(E+c−E−c)*100

TABLE 4 Percentage of of IFN gamma secretion after allogenic stimulationand treatment with anti-PD-1 antibody in comparison to effect ofrecombinant human IL-2 treatment (20 (EU/ml) (=100% increase) aspositive control Concentration (μg/ml) 1:12 1:120 1:1200 Effect in MLRPD1-0050 44 136 96 33 +++ PD1-0069 60 76 71 55 +++ PD1-0073 43 103 63 38++ PD1-0078 64 99 72 21 ++

Several PD1 blocking antibodies PD1-0050, PD1-0069, PD1-0073, PD1-0078,PD1-0098, PD1-0102, PD1-0103 demonstrated strong immune modulatingactivity by enhancing secretion of interferon gamma (IFNγ) (data notshown for all antibodies).

3B) In a further experiment chimeric PD1-0103 (human IgG1 isotype withmutations L234A, L235A and P329G (EU index of Kabat)) was evaluated.Blockade of PD1 with chimeric PD1-0103 strongly enhances IFN-gammasecretion by allogenic stimulated primary human T cells. ChimericPD1-0103 was more potent than reference anti-PD1 antibodies. Forcomparison the reference anti-PD1 antibodies comprising the VH and VLdomains of either nivolumab (also known as MDX5C4 or MDX-1106) andpembrolizumab (also known as MK-3475 or Org 1.09A) were synthesized andcloned with backbones of human IgG1 (with mutations L234A. L235A andP329G (EU index of Kabat)) were used.

3C) In additional experiments the immune modulating activity of thehumanized variants of anti-PD-1 antibody PD1-0103 (humanized antibodiesPD1-0103-0312, PD1-0103-0314, in FIG. 2 and FIG. 3, see also Example 9below) the a) IFNγ release (secretion) b) TNF-alpha release (secretion)was evaluated in MLR as described above. The effect of the chimericPD1-0103 antibody and its humanized versions were compared to thereference anti-PD1 antibodies comprising the VH and VL domains of eithernivolumab (also known as MDX5C4 or MDX-1106) and pembrolizumab (alsoknown as MK-3475 or Org 1.09A) with backbones of human IgG1 (withmutations L234A, L235A and P329G (EU index of Kabat)). After 6 days ofMLR culture 50 μl of supernatant was taken and multiple cytokines weremeasured in a single culture using Bio-Plex Pro™ Human Cytokine Th1/Th2Assay (Bio-Rad Laboratories Inc.). (data not shown for all cytokines).The chimeric PD1-0103 antibody and its humanized versions (PD1-0103_0312and PD1-0103_0314) were more potent compared to the reference anti-PD1antibodies in enhancing the T cell activation and IFN-gamma secretion.Furthermore, the chimeric PD1-0103 antibody and its humanizationvariants increased tumor necrosis factor alpha (TNF alpha) and IL-12secretion by antigen presenting cells and encance capacity ofmonocytes/macrophages or antigen presenting cells to stimulate a T cell.

Example 4 Effect of Anti-PD-1 Blockade on Cytotoxic Granzyme B Releaseand IFN-γ Secretion by Human CD4 T Cells Cocultured with AllogeneicMature Dendritic Cells

To further investigate the effect of anti-PD-1 treatment in anallogeneic setting we developed an assay in which freshly purified CD4 Tcells are cocultured for 5 days in presence of monocyte-derivedallogeneic mature dendritic cells (mDCs). Monocytes were isolated fromfresh PBMCs one week before through plastic adherence followed by theremoval of the non-adherent cells. We then generated immature DCs fromthe monocytes by culturing them for 5 days in media containing GM-CSF(50 ng/ml) and IL-4 (100 ng/ml). To induce iDCs maturation, we addedTNF-α, IL-1β and IL-6 (50 ng/ml each) to the culturing media for 2additional days.

We then assessed DCs maturation by measuring their surface expression ofMajor Histocompatibility Complex Class II (MHCII), CD80, CD83 and CD86thorugh flow cytometry (LSRFortessa, BD Biosciences).

On the day of the minimal mixed lymphocyte reaction (mMLR), CD4 T cellswere enriched via a microbead kit (Miltenyi Biotec) from 10⁸ PBMCsobtained from an unrelated donor. Prior culture, CD4 T cells werelabeled with 5 μM of Carboxy-Fluorescein-Succinimidyl Esther (CFSE). 10⁵CD4 T cells were then plated in a 96 well plate together with matureallo-DCs (5:1) in presence or absence of blocking anti-PD1 antibody(either PD1-0103, chimeric PD1-0103, or humanized antibodiesPD1-0103-0312, PD1-0103-0313, PD1-0103-0314, PD1-0103-0315, abbreviatedas 0312, 0313, 0314, 0315), at the concentration of 10 μg/ml if notdifferently indicated in the figures.

Five days later the cell-culture supernatants were collected and used tomeasure the IFN-γ levels by ELISA (R&D systems. The cells were left at37° C. for additional 5 hours in presence of Golgi Plug (Brefeldin A)and Golgi Stop (Monensin). The cells were then washed, stained on thesurface with anti-human CD4 antibody and the Live/Dead fixable dye Aqua(Invitrogen) before being fixed/permeabilized with Fix/Perm Buffer (BDBioscience). Intracellular staining was performed for Granzyme B (BDBioscience), IFN-γ and IL-2 (both from eBioscience).

All humanized variants PD1-0103 (humanized antibodies PD1-0103-0312,PD1-0103-0313, PD1-0103-0314, PD1-0103-0315, abbreviated as 0312, 0313,0314, 0315) were found to be equally good in enhancing granzyme B andinterferon gamma (data not shown).

Example 5 Chimeric PD1 Antibody Derivatives

Chimeric PD1 antibodies were generated by amplifying the variable heavyand light chain regions of the anti-PD1 mouse antibodies PD1-0098,PD1-0103 via PCR and cloning them into heavy chain expression vectors asfusion proteins with human IgG1 backbones/human CH1-Hinge-CH2-CH3 withmutations L234A, L235A and P329G (EU index of Kabat)) (Leucine 234 toAlanine, Leucine 235 to Alanine, Proline 329 to Glycine) abrogatingeffector functions and light chain expression vectors as fusion proteinsto human C-kappa. LC and HC Plasmids were then cotransfected into HEK293and purified after 7 days from supertnatants by standard methods forantibody purification. The chimeric PD1-antibodies were renamed chimericchiPD1-0098 (chiPD1-0098) and chimeric PD1-0103 (chiPD1-0103). Forcomparison the reference anti-PD1 antibodies comprising the VH and VLdomains of either nivolumab (also known as MDX-5C4 or MDX-1106) andpembrolizumab (also known as MK-3475 or Org 1.09A) were synthesized andcloned with backbones of human IgG1 (with mutations L234A, L235A andP329G (EU index of Kabat)) were used.

Example 6 Generation, Expression and Purification of Humanized Variantsof Anti-PD1 Antibody PD-0103 (huMab PD-0103) and CharacterizationHumanization of the VH and VL Domains of Murine Anti-PD1 Antibody 0103

Based upon the amino acid sequence of the murine VH and VL domains ofmurine anti-PD1 antibody PD1-0103 (SEQ ID NO: 7 and 8), humanizedanti-anti-PD1 antibody variants were generated.

The humanized VH-variant is based on the human germline IMGT_hVH_3_23 incombination with the human J-element germline IGHJ5-01 with severalmutations. (resulting in SEQ ID NO: 9).

The humanized variants of VL are based on the human germlinesIMGT_hVK_4_1, IMGT_hVK_2_30, IMGT_hVK_3_11 and IMGT_hVK_1_39 incombination with the human J-element germline IGKJ1-01. Differentmuations resulted in humanized variants of SEQ ID NO: 10 to SEQ ID NO:13.

The humanized amino acid sequences for heavy and light chain variableregions of PD1-0103 were backtranslated in to DNA and the resulting cNDAwere synthesized (GenArt) and then cloned into heavy chain expressionvectors as fusion proteins with human IgG1 backbones/humanCH1-Hinge-CH2-CH3 with LALA and PG mutations (Leucine 234 to Alanine,Leucine 235 to Alanine. Proline 329 to Glycine) abrogating effectorfunctions or into light chain expression vectors as fusion proteins tohuman C-kappa. LC and HC Plasmids were then cotransfected into HEK293and purified after 7 days from supertnatants by standard methods forantibody purification. The resulting humanized PD1-antibodies named asfollows:

TABLE 5 VH and VL sequences of humanized variant antibodies of PD1-0103Humanized antibodies humanized variant of humanized variant of ofPD1-0103 VH/SEQ ID NO: VL/SEQ ID NO: PD1-0103-0312 SEQ ID NO: 9 SEQ IDNO: 10 PD1-0103-0313 SEQ ID NO: 9 SEQ ID NO: 11 PD1-0103-0314 SEQ ID NO:9 SEQ ID NO: 12 PD1-0103-0315 SEQ ID NO: 9 SEQ ID NO: 13

Humanized PD1-0103 antibody variants and parental chimeric PD1-0103 werecharacterized as described above. Results are shown in Table 6.

TABLE 6 Summary of results for humanized PD1-0103 antibody variants andparental chimeric PD1-0103 chimeric PD-0103- PD-0103- PD-0103- PD-0103-Assay PD1-0103 0312 0313 0314 0315 Affinity 2.0/0.8 1.5/1.8 1.9/2.31.6/1.5 1.7/1.5 K_(D 37°) _(C.) [nM] ELISA 0.2 0.1 0.07 0.07 0.06 EC₅₀[nM] CHO-PD1 EC₅₀ + + + + + IC₅₀ PD-L1, 1.35 tbd tbd tbd tbd 2 [nM]Mixed +++ +++ +++ ++++ ++ Lymphocyte Reaction assay cynomolgus + 0.080.06 0.05 0.04 crossreactivity (EC₅₀ [nm])

The humanized variant PD-0103-0312 is termed aPD1 antibody clonePD1-0376 in the following.

Example 7 Generation of Anti-LAG3 Antibodies Immunization of Rabbits

Roche proprietary transgenic rabbits expressing a humanized antibodyrepertoire were immunized with LAG3 expressing plasmid DNA.

A set of 3 rabbits was immunized genetically, using a plasmid expressionvector coding for full-length human LAG3(15352_pIntronA_fl-hLag3_DNA-IMS), by intradermal application of 400 ugvector DNA, followed by Electroporation (5 square pulses of 750 V/cm,duration 10 ms, interval 1 s). Rabbits received 7 consecutiveimmunizations at days 0, 14, 28, 49, 70, 98 and 126. Blood (10% ofestimated total blood volume) was taken at days 35, 77, 105 and 133.Serum was prepared, which was used for titer determination by ELISA (seebelow), and peripheral mononuclear cells were isolated, which were usedas a source of antigen-specific B cells in the B cell cloning processbelow.

Determination of Serum Titers (ELISA)

Human recombinant LAG3 protein was immobilized on a 96-well NUNCMaxisorp plate at 2 ug/ml, 100 ul/well, in PBS, followed by: blocking ofthe plate with 2% Crotein C in PBS, 200 ul/well; application of serialdilutions of antisera, in duplicates, in 0.5% Crotein C in PBS, 100ul/well; detection with either (1) HRP-conjugated donkey anti-rabbit IgGantibody (Jackson Immunoresearch/Dianova 711-036-152: 1/16 000), or (2)HRP-conjugated rabbit anti-human IgG antibody (Pierce/Thermo Scientific31423; 1/5000), or (3) biotinylated goat anti-human kappa antibody(Southern Biotech/Biozol 2063-08, 1/5000) and streptavidin-HRP; eachdiluted in 0.5% Crotein C in PBS, 100 ul/well. For all steps, plateswere incubated for 1 h at 37° C. Between all steps plates were washed 3times with 0.05% Tween 20 in PBS. Signal was developed by addition of BMBlue POD Substrate soluble (Roche), 100 ul/well; and stopped by additionof 1 M HCl, 100 ul/well. Absorbance was read out at 450 nm, against 690nm as reference. Titer was defined as dilution of antisera resulting inhalf-maximal signal.

Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMC)

Blood samples were taken of immunized transgenic rabbits. EDTAcontaining whole blood was diluted twofold with 1×PBS (PAA, Pasching,Austria) before density centrifugation using lympholyte mammal(Cedarlane Laboratories, Burlington, Ontario, Canada) according to thespecifications of the manufacturer. The PBMCs were washed twice with1×PBS.

EL-4 B5 Medium

RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS(Hyclone, Logan, Utah, USA), 2 mM Glutamin, 1% penicillin/streptomycinsolution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES(PAN Biotech, Aidenbach, Germany) and 0.05 mM b-mercaptoethanole (Gibco,Paisley, Scotland) was used.

Coating of Plates with Protein Antigen

Sterile cell culture 6-well plates were coated with human LAG3 ECDconjugated to a human Fc part (2 μg/ml) in carbonate buffer (0.1 Msodium bicarbonate, 34 mM Disodiumhydrogencarbonate. pH 9.55) over nightat 4° C. Plates were washed in sterile PBS three times before use.

Depletion of Cells

(a) Sterile 6-well plates (cell culture grade) covered with a confluentmonolayer of CHO cells were used to deplete macrophages/monocytesthrough unspecific adhesion as well as unspecifically bindinglymphocytes.

(b) Blank sterile 6-well plates (cell culture grade) were used todeplete macrophages and monocytes and other cells through unspecificadhesion.

Half of the PBMC sample was used for (a) and half for (b).

Each well was filled at maximum with 4 ml medium and up to 6×106 PBMCsfrom the immunized rabbit and allowed to bind for 1 h at 37° C. in theincubator. The cells in the supernatant (peripheral blood lymphocytes(PBLs)) were used for the antigen panning step.

Enrichment of B Cells on LAG3 Antigen

Protein Antigen: 6-well tissue culture plates coated with LAG3-ECD-huFcprotein were seeded with up to 6×10⁶ PBLs per 4 ml medium from thedepletion steps using the blank 6-well plate and allowed to bind for 1 hat 37° C. in the incubator. Non-adherent cells were removed by carefullywashing the wells 1-2 times with 1×PBS. The remaining sticky cells weredetached by trypsin for 10 min at 37° C. in the incubator. Trypsinationwas stopped with EL-4 B5 medium. The cells were kept on ice until theimmune fluorescence staining.

Cell surface antigen: 6-well tissue culture plates covered with amonolayer of human LAG3-positive CHO cells were seeded with up to 6×10⁶PBLs per 4 ml medium from the depletion steps using the CHO-covered6-well plate and allowed to bind for 1 h at 37° C. in the incubator.Non-adherent cells were removed by carefully washing the wells 1-2 timeswith 1×PBS. The remaining sticky cells were detached by trypsin for 10min at 37° C. in the incubator. Trypsination was stopped with EL-4 B5medium. The cells were kept on ice until the immune fluorescencestaining.

Immune Fluorescence Staining and Flow Cytometry

The anti-IgG FITC (AbD Serotec, Düsseldorf, Germany) and the anti-huCkPE (Dianova, Hamburg, Germany) antibody was used for single cellsorting. For surface staining, cells from the depletion and enrichmentstep were incubated with the anti-IgG FITC and the anti-huCk PE antibodyin PBS and incubated for 45 min in the dark at 4° C. After staining thePBMCs were washed two fold with ice cold PBS. Finally the PBMCs wereresuspended in ice cold PBS and immediately subjected to the FACSanalyses. Propidium iodide in a concentration of 5 μg/ml (BD Pharmingen,San Diego, Calif., USA) was added prior to the FACS analyses todiscriminate between dead and live cells. A Becton Dickinson FACSAriaequipped with a computer and the FACSDiva software (BD Biosciences, USA)were used for single cell sort.

B-Cell Cultivation

The cultivation of the rabbit B cells was performed by a methoddescribed by Seeber et al. (S Seeber et al. PLoS One 9 (2), e86184, 2014Feb. 4). Briefly, single sorted rabbit B cells were incubated in 96-wellplates with 200 μl/well EL-4 B5 medium containing Pansorbin Cells(1:100000) (Calbiochem (Merck), Darmstadt, Deutschland), 5% rabbitthymocyte supernatant (MicroCoat, Bernried, Germany) andgamma-irradiated murine EL-4 B5 thymoma cells (5×10e5 cells/well) for 7days at 37° C. in the incubator. The supernatants of the B-cellcultivation were removed for screening and the remaining cells wereharvested immediately and were frozen at −80° C. in 100 μl RLT buffer(Qiagen, Hilden, Germany).

Isolation of V-Domains of LAG3 Antibodies

PCR Amplification of V-Domains

Total RNA was prepared from B cells lysate (resuspended in RLTbuffer-Qiagen-Cat. No 79216) using the NucleoSpin 8/96 RNA kit(Macherev&Nagel; 740709.4, 740698) according to manufacturer's protocol.RNA was eluted with 60 μl RNase free water. 6 μl of RNA was used togenerate cDNA by reverse transcriptase reaction using the SuperscriptIII First-Strand Synthesis SuperMix (Invitrogen 18080-400) and an oligodT-primer according to the manufatures's instructions. All steps wereperformed on a Hamilton ML Star System. 4 μl of cDNA were used toamplify the immunoglobulin heavy and light chain variable regions (VHand VL) with the AccuPrime Supermix (Invitrogen 12344-040) in a finalvolume of 50 μl using the primers rbHC.up and rbHC.do for the heavychain and BcPCR_FHLC_leader.fw and BcPCR_huCkappa.rev for the lightchain (Table 7). All forward primers were specific for the signalpeptide (of respectively VH and VL) whereas the reverse primers werespecific for the constant regions (of respectively VH and VL). The PCRconditions for the RbVH were as follows: Hot start at 94° C. for 5 min;35 cycles of 20 s at 94° C., 20 s at 70° C., 45 s at 68° C., and a finalextension at 68° C. for 7 min. The PCR conditions for the HuVL were asfollows: Hot start at 94° C. for 5 min; 40 cycles of 20 s at 94° C., 20s at 52° C., 45 s at 68° C., and a final extension at 68° C. for 7 min.

TABLE 7 SEQ ID NO: 76 AAGCTTGCCACCATGGAGACTGGGCTGCGCTGG rbHC.up CTTCSEQ ID NO: 77 CCATTGGTGAGGGTGCCCGAG rbHCf.do SEQ ID NO: 78ATGGACATGAGGGTCCCCGC BcPCR_FHLC_ leader.fw SEQ ID NO: 79GATTFCAACTGCTCATCAGATGGC BcPCR_huCkappa_ rev

8 μl of 50 μl PCR solution were loaded on a 48 E-Gel 2% (InvitrogenG8008-02). Positive PCR reactions were cleaned using the NucleoSpinExtract II kit (Macherey & Nagel; 740609250) according to manufacturer'sprotocol and eluted in 50 μl elution buffer. All cleaning steps wereperformed on a Hamilton ML Starlet System.

Recombinant Expression of Rabbit Monoclonal Bivalent Antibodies

For recombinant expression of rabbit monoclonal bivalent antibodies,PCR-products coding for VH or VL were cloned as cDNA into expressionvectors by the overhang cloning method (R S Haun et al., Biotechniques(1992) 13, 515-518; M Z Li et al., Nature Methods (2007) 4, 251-256).The expression vectors contained an expression cassette consisting of a5′ CMV promoter including intron A, and a 3′ BGH poly adenylationsequence. In addition to the expression cassette, the plasmids containeda pUC18-derived origin of replication and a beta-lactamase geneconferring ampicillin resistance for plasmid amplification in E. coli.Three variants of the basic plasmid were used: one plasmid containingthe rabbit IgG constant region designed to accept the VH regions whilecontaining human kappa LC constant region to accept the VL regions.Linearized expression plasmids coding for the kappa or gamma constantregion and VL/VH inserts were amplified by PCR using overlappingprimers. Purified PCR products were incubated with T4 DNA-polymerasewhich generated single-strand overhangs. The reaction was stopped bydCTP addition.

In the next step, plasmid and insert were combined and incubated withrecA which induced site specific recombination. The recombined plasmidswere transformed into E. coli. The next day the grown colonies werepicked and tested for correct recombined plasmid by plasmid preparation,restriction analysis and DNA-sequencing.

For antibody expression, the isolated HC and LC plasmids weretransiently co-transfected into HEK293 cells and the supernatants wereharvested after 1 week.

Example 8 Characterization of Anti-LAG3 Antibodies

TABLE 8 Summary of Characterization of different anti-LAG3 Antibodiesanti-Lag3 aLAG3 aLAG3 aLAG3 aLAG3 aLAG3 MDX- BMS- MDX-26H10 HumanizedBAP antibodies (0403) (411) (414) (416) (417) 25F7(25F7) 986016 (26H10)050 (LAG525) K_(D) [M] tbd tbd 4.63 2.82 tbd tbd tbd tbd tbd monovalentE−10 E−11 bivalent tbd tbd tbd tbd tbd tbd tbd tbd tbd kd [1/s] 5.003.87 1.95 2.21 9.48 3.86 3.99 E−06 E−05 E−04 E−04 E−05 E−04 E−04 EpitopeE3 E3 E3 E2b E3 E5 E5 E4 E2c Bin (D1−loop) MHCII/ 0.9 0.8 0.9 0.9 0.90.8/0.6 /0.4 0.9/0.6 /1.0 ELISA IC₅₀ [nM] CHO-cell 30.9 41.3 48.1 37.227.8 75 ELISA inflexion point [ng/ml]

ELISA for Human Lag3

Nunc maxisorp plates (Nunc 464718) were coated with 25 μl/wellrecombinant Human LAG-3 Fc Chimera Protein (R&D Systems, 2319-L3) at aprotein concentration of 800 ng/ml and incubated at 4° C. overnight orfor 1 h at room temperature. After washing (3×90 μl/well withPBST-buffer) each well was incubated with 90 μl blocking buffer (PBS+2%BSA+0.05% Tween 20) for 1 h at room temperature. After washing (3×90μl/well with PBST-buffer) 25 μl anti-Lag3 samples at a concentration of1-9 μg/ml (1:3 dilutions in OSEP buffer) were added and incubated 1 h atRT. After washing (3×90 μl/well with PBST-buffer) 25 μl/well goatanti-Human Ig κ chain antibody-HRP conjugate (Milipore, AP502P) wasadded in a 1:2000 dilution and incubated at RT for 1 h. After washing(3×90 μL/well with PBST-buffer) 25 μl/well TMB substrate (Roche,11835033001) was added and incubated for 2-10 min. Measurement tookplace on a Tecan Safire 2 instrument at 370/492 nm.

Cell-Surface Lag3 Binding ELISA

25 μl/well of Lag3 cells (recombinant CHO cells expressing Lag3, 10000cells/well) were seeded into tissue culture treated 384-well plates(Corning, 3701) and incubated at 37° C. for one or two days. The nextday after removal of medium, 25 μl anti-Lag3 samples (1:3 dilutions inOSEP buffer, starting at a concentration of 6-40 nM) were added andincubated for 2 h at 4° C. After washing (1×90 μl in PBST) cells werefixed by addition of 30 μl/well glutaraldehyde to a final concentrationof 0.05% (Sigma Cat.No: G5882), 10 min at room temperature. Afterwashing (3×90 μl/well with PBST-buffer) 25 μl/well goat anti-Human Ig κchain antibody-HRP conjugate (Milipore, AP502P) was added in a 1:1000dilution and incubated at RT for 1 h. After washing (3×90 pt/well withPBST-buffer) 25 μl/well TMB substrate (Roche, 11835033001) was added andincubated for 6-10 min. Measurement took place on a Tecan Satire 2instrument at 370/492 nm.

SPR (Biacore) Characterization of Anti-LAG3 Antibodies

A surface plasmon resonance (SPR) based assay has been used to determinethe kinetic parameters of the binding between anti-Lag3 antibodies inbivalent format or as monovalent Fab fragments and human Fc tagged humanLag3 extra cellular domains (ECDs) at 25° C.

Therefore two flow cells of a C1 biosensor chip were prepared in aBiacore T200 by immobilizing neutravidin, diluted to 25 μg/ml in acetatebuffer pH 4.5, onto it using the ‘immobilization wizard’. This yieldedin immobilization levels of around 1900 RU. Then, CaptureSelect™ BiotinAnti-IgG-Fc (Human) Conjugate was bound to the neutravidin, using a 20μg/ml dilution in running buffer (HBS-EP+, GE Healthcare).

The method itself consisted of four commands per cycle. First command:capturing of ˜46 RU of huLag3-Fc (20 s, 10 μl/min). Second command:sample injection for 120 s followed by a 1200 s long dissociation at aflow speed of 30 μl/min. Third and fourth command: regeneration byinjecting Glycine-HCl pH 1.5 for 30 seconds. A dilution series (3.13nM-200 nM, two-fold dilutions in running buffer) of each antibody Fabfragment and additional blank cycles were then measured using thepreviously described method. The Biacore T200 Evaluation Software wasthen utilized to gain kinetic values by applying a 1:1 Langmuir fit withthe Rmax fit parameter set to ‘local’ since the capture levels were notperfectly reproducible. Results (K_(D) values and kd values) are shownin Table 8.

Epitope Mapping

Epitope binning was performed using a surface plasmon resonance (SPR)based assay. Therefore aLag3 binders were bound to huLag3 on a BiacoreT200 instrument. Then the accessibility of other binders to thepreviously formed aLag3 binder-huLag3 complex was assessed.

A SA CAP Kit (GE Healthcare) was used to carry out this assay. If notdescribed otherwise, the assay was done according to the SA CAP Kitmanual. The run included only one cycle type. After hybridization, a 10nM dilution of biotinylated, huFc-tagged huLag3 was allowed to bind tothe streptavidin on the sensor chip for 20 s at a flow rate of 10μl/min. Then a first 200 nM sample diluted in running buffer wasinjected for 180 s at a flow rate of 30 μl/min and immediately followedby a second sample under the same conditions. The surface was thenregenerated.

The samples were then assigned to different epitope groups with similarcompetition patterns. A first rough categorization was done, based onthe relative response of the second injection using a threshold of 6.1RU, which was just above the highest value observed when a binder wasinjected as first and second sample. All values and decisions werefinally validated by visual inspection of the sensorgrams.

Results are shown in Table 8. Three major epitope patterns (E1, E2 andE3) were identified. Since aLag3-0416 and humanized BAP 050 share thesame group but do not completely inhibit each other, they were assignedto subgroups E2b and E2c.

Binding of Anti-Lag3 Antibodies from Tg Rabbits to Recombinant Cyno Lag3Positive HEK Cells

In addition to the binding analysis using HEK cells recombinantlyexpressing human Lag3 on the surface, binding to cynomolgusLag3-positive HEK cells was also evaluated. For this experiment, frozenHEK293F cells, previously transiently transfected with cyno-LAG-3, werethawed, centrifuged and resupplemented in PBS/2% FBS. 1.5×10⁵ cells/wellwere seeded into 96-well plates. Anti-Lag3 antibodies wered added to afinal normalized concentration of 10 g/ml. For referencing and ascontrols, autofluorescence and positive control (Medarex 25F7) as wellas isotype control (huIgGI from Sigma, cat.no. #I5154, data not shown)antibodies were prepared and measured in the experiment. HEK cells wereincubated with indicated antibodies for 45 min on ice, washed twice with200 μl ice-cold PBS buffer containing 2% FBS, before secondary antibody(APC-labelled goat anti-human IgG-kappa, Invitrogen, cat.no.#MH10515)was added (1:50 diluted in FACS-Puffer/well) and further incubated for30 min on ice. Cells were again washed twice with 200 μl ice-cold PBS/2%FBS buffer before samples were finally resuspended in 150 μl FACS bufferand binding was measured on FACS CANTO-II HTS Module.

Results:

Shown in the below table is the binding and cross-reactivity ofdifferent anti-Lag3 antibodies to HEK293 cells expressing cynoLAG3,binding either given in % positive cells or the GeoMean of the signalintensity.

TABLE 9 Binding of different anti-LAG3 Antibodies to recombinant cynoLag3 positive HEK cells LAG3 antibody % pos. GeoMean Reference LAG3antibody MDX25F7 41.2 3062 aLAG3(0411) 88.6 11007 aLAG3(0414) 81.6 9169aLAG3(0416) 67.9 4221 aLAG3(0417) 75.9 7115 aLAG3(0403) 82.0 7457Binding of Anti-Lag3 Antibodies from Tg Rabbits to (Activated)Cynomolgus PBMC/T Cells Expressing Lag3

After binding to recombinant Lag3 protein and Lag3 expressedrecombinantly on mammalian cells, binding to Lag3 expressed on activatedcynomolgus T cells was also assessed.

The binding characteristics of the newly generated anti-Lag3 antibodies(derived from Roche's transgenic rabbits) to Lag3 expressed on the cellsurface of cynomolgus T cells or PBMC was confirmed by FACS analysis.While Lag3 is not expressed on naïve T cells it is upregulated uponactivation and/or on exhausted T cells. Thus, cynomolgus peripheralblood mononuclear cells (PBMC) were prepared from fresh cynomolgus bloodand were then activated by CD3/CD28 pre-treatment (1 μg/ml) for 2-3days. Activated cells were subsequently analyzed for Lag3 expression:Briefly, 1-3×10⁵ activated cells were stained for 30-60 min on ice withindicated anti-Lag3 antibodies and respective control antibodies at 10μg/ml final concentration. The bound anti-Lag3 antibodies were detectedvia fluorochrome-conjugated anti-human IgG or an anti-rabbit IgGsecondary antibodies. After staining, cells were washed two times withPBS/2% FCS and analyzed on a FACS Fortessa (BD).

Result:

The following table summarizes the percentage of Lag3 positive cellswithin activated cynomolgus PBMC.

TABLE 10 Binding of different anti-LAG3 Antibodies to (activated)cynomolgus PBMCs/T cells expressing Lag3 % positive cyno cells (PBLs)after Anti-Lag3/ctrl Antibodies CD3/CD28 activation only 2nd Ab (hu)7.62 DP47 (human isotype) 9.19 Reference LAG3 antibody 22.1 (MDX25F7)Reference LAG3 antibody 18.6 BMS-986016 Reference LAG3 antibody 50.7(humanized BAP050(LAG525)) only 2nd Ab (rb) 5.26 aLAG3(0403) 44.2aLAG3(0411) 46.6 aLAG3(0414) 43.0 aLAG3(0416) 38.9 aLAG3(0417) 35.3

On activated cynomolgus T cells all of the rabbit anti-Lag3 antibodiesdemonstrated a significant binding to Lag3⁺ cells. Hereby, all newlygenerated antibodies showed an increased percentage of positive cellscompared to human anti-Lag3 reference antibodies (e.g. such as MDX25F7,BMS-986016).

Inhibition of LAG-3 Binding to MHC-II Expressed on Human A375 TumorCells (by ELISA)

25 μl/well of A375 cells (10000 cells/well) were seeded into tissueculture treated 384-well plates (Corning, 3701) and incubated at 37° C.overnight. Anti-Lag3 antibodies were pre-incubated for 1 h withbiotinylated-Lag3 (250 ng/ml) in cell culture medium in 1:3 dilutionsstarting at 3 μg/ml antibody-concentration. After removal of medium fromthe wells with the seeded cells, 25 μl of the antibody-Lag3pre-incubated mixtures were transferred to the wells and incubated for 2h at 4° C. After washing (1×90 μl in PBST) cells were fixed by additionof 30 μl/well glutaraldehyde to a final concentration of 0.05% (SigmaCat.No: G5882), 10 min at room temperature. After washing (3×90 μl/wellwith PBST-buffer) 25 μl/well Poly-HRP40-Streptavidin (Fitzgerald,65R-S104PHRPx) was added in a 1:2000 or 1:8000 dilution and incubated atRT for 1 h. After washing (3×90 μl/well with PBST-buffer) 25 μl/well TMBsubstrate (Roche, #11835033001) was added and incubated for 2 to 10 min.Measurement took place on a Tecan Safire 2 instrument at 370/492 nm.

Inhibition of LAG-3 Binding to MHC-II Expressed on Human A375 TumorCells (by FACS Analysis)

Assay Principle:

To study the antagonistic function of the anti-Lag3 antibodies, anMHCII:Lag3 competition assay was conducted. MHCII⁺ human A375 cells werestained with inhouse generated biotinylated Lag3:Fc fusion protein withor without pre-incubation with anti-Lag3 antibodies. This analysis wasstudied in a FACS competition experiment: A375 cells (ATCC, #CRL-1619)were cultured for 2-3 passages in EM Eagle's medium supplemented withEBSS (PAN, cat.no. #P04-00509), 10% FBS, 2 mM L-Glutamin, 1×NEAA and 1×Sodium Pyruvate. All antibodies, were diluted in FACS buffer to a finalconcentration of 20 μg/ml in 25 μl (in 96 well U-bottom plates). 25 μlof inhouse generated, biotinvlated recombinant LAG-3:Fc fusion proteinwas added to a final concentration of 10 μg/ml either to medium or toanti-Lag3 antibodies or controls and were pre-incubated for 30 min atroom temperature. A375 cells were washed with PBS and adjusted to 3×10⁶cells/ml in PBS. 100 μl were seeded per well in a 96 well V-bottomplate. Plates were centrifuged and supernatant was removed. Then thepre-incubated LAG-3:Fc fusion protein/antibody mix (50 l/well) was addedto the cells and incubated for 1 h at room temperature. After this,cells were washed with 200 μl FACS buffer. For detection of biotinylatedLag3:Fc protein bound to cellular MHCII, an APC-conjugated goatanti-Biotin antibody was used at 3 μl/sample (Miltenyi Biotec, cat.no.#130-090-856) and incubated for additional 10-15 mins. After staining,cells were again washed and then transferred in 150 μl FACS buffer(PBS/2% FBS) to a U-bottom plate and analyzed on a FACS Canto-II usingan HTS module.

Two anti-Lag3 antibodies (clones 25F7 and 26H10; Medarex) served aspositive controls and a human IgG1 (Sigma. cat.no. #15154) asappropriate isotype control. All antibodies were used at 10 μg/ml finalconcentration.

Results:

Shown in the below table is the result of the FACS analysisdemonstrating the percent inhibition of the Lag3 protein binding toMHC-II on cells (calculated as the reduced binding signal in referenceto the maximal value in the absence of a blocking antibody).

TABLE 11 Binding of different anti-LAG3 Antibodies to (activated)cynomolgus PBMC/T cells expressing Lag3 aLAG3 antibody % InhibitionaLAG3(0403) 34.9 aLAG3(0414) 67.3 aLAG3(0411) 45.6 aLAG3(0416) 68.6aLAG3(0417) 59.1 Reference MDX25F7 70.0 Reference MDX26H10 71.7 Isotypecontrol −2.9 No mAb 0.0

These data support a functional interplay with Lag3 and blockade of thecellular interaction of all tested antibodies.

Neutralizing Potency of the Novel Anti-Lag3 Antibodies in a StandardLAG3 Blockade Bio/Reporter Assay

To test the neutralizing potency of the novel anti-Lag3 antibodies inrestoring a suppressed T cell response in vitro, a commerciallyavailable reporter system was used. This system consists of Lag3⁺ NFATJurkat effector cells (Promega, cat. no. #CS194801), MHC-II⁺ Raji cells(ATCC, #CLL-86), and a super-antigen. In brief, the reporter system isbased on three steps: (1) superantigen-induced NFAT cell activation, (2)inhibition of the activating signal mediated by the inhibitinginteraction between MHCII (Raji cells) and Lag3⁺ NFAT Jurkat effectorcells, and (3) recovery of the NFAT activation signal byLag3-antagonistic/neutralizing aVH-Fc fusion constructs.

For this experiment, Raji and Lag-3⁺ Jurkat/NFAT-luc2 effector T cellswere cultured as described by the provider. Serial dilutions (40pg/ml-50 μg/ml) of several anti-Lag3 and reference antibodies wereprepared in assay medium (RPMI 1640 (PAN Biotech. cat.no. #P04-18047),1% FCS) in flat, white bottom 96-well culture plates (Costar,cat.no.#3917). 1×10⁵ Lag3⁺ NFAT-Jurkat cells/well) were added to theantibody solution. After this step, 2.5×10⁴ Raji cells/well were addedto the Jurakt cell/antibody mix as well as 50 ng/ml final concentrationof the SED super-antigen (Toxin technology, cat.no. DT303). After anincubation of six hrs at 37° C. and 5% CO₂, Bio-Glo substrate (Promega,#G7940) was warmed up to room temperature and 75 μl were added per well,incubated for 5-10 min before the overall luminescence was measured at aTecan Infinite reader according to the kit's manufacturer'srecommendation.

Shown in the table is the restoration of a MHCII/Lag3-mediatedsuppression of the NFAT luciferase signal by different anti-Lag3antibodies upon SED stimulation (given as EC₅₀ values):

TABLE 12 Results with different anti-LAG3 Antibodies in the standardLAG3 Blockade Bio/Reporterassay EC₅₀ [nM] in Jurkat LAG3 + SED + RajiAnti-LAG3 1st assay 2nd assay 3rd assay Reference MDX25F7 7.8/5.9 8.6n.t. Reference n.t. 9.6 n.t. BMS-986016 Reference humanized n.t. 22.6n.t. BAP050(LAG525) Lag3 IgG-Fc n.t. no effect n.t. aLAG3(0411) 1.1 1.0n.t. aLAG3(0414) 1.1 1.0 1.8 aLAG3(0416) 3.1 2.5 3.5 aLAG3(0417) 1.0n.t. n.t. n.t. molecules not tested in this experiement

Example 9 Functional Characterization of Anti-LAG3 Antibodies

Table 13 summarizes the biological activity and effects of differentanti-LAG3 antibodies (alone or in combination with anti-PD1 antibodies)in different assays as described herein.

TABLE 13 Summary of biologival activity of different anti-LAG3Antibodies (alone or in combination with anti-PD1 antibodies) Anti-Anti- Anti- Anti- Anti- Ref. 2 Lag3 Lag3 Lag3 Lag3 Lag3 Ref. 1 humanizedAssay aLAG3 aLAG3 aLAG3 aLAG3 aLAG3 BMS BAP050 type (0403) (0411) (0414)(0416) (0417) 986016 (LAG525) mMLR + − +++ ++ + − ++ (GrzB) mMLR − − + +++ + ++ (IL-2) CD4 + AR +++ +++ + + H77 Treg- +++ + − + suppression(GrzB) Treg- +++ ++ + + suppression (IFN-γ) Melanoma +++ patient PBMCsEffect of PD-1 and LAG-3 Blockade on Cytotoxic Granzyme B Release andIL-2 Secretion by Human CD4 T Cells Cocultured with Allogeneic MatureDendritic Cells

To screen anti-LAG-3 blocking antibodies in combination with anti-PD-1in an allogeneic setting an assay was developed in which freshlypurified CD4 T cells are cocultured for 5 days in presence ofmonocyte-derived allogeneic mature dendritic cells (mDCs). Monocyteswere isolated from fresh PBMCs one week before through plastic adherencefollowed by the removal of the non-adherent cells. Immature DCs werethen generated from the monocytes by culturing them for 5 days in mediacontaining GM-CSF (50 ng/ml) and IL-4 (100 ng/ml). To induce iDCsmaturation, TNF-alpha, IL-1beta and IL-6 (50 ng/ml each) were added tothe culturing media for 2 additional days. DCs maturation was thenassessed by measuring their surface expression of MajorHistocompatibility Complex Class II (MHCII), CD80, CD83 and CD86 thorughflow cytometry (LSRFortessa. BD Biosciences).

On the day of the minimal mixed lymphocyte reaction (mMLR), CD4 T cellswere enriched via a microbead kit (Miltenyi Biotec) from 10⁸ PBMCsobtained from an unrelated donor. Prior culture, CD4 T cells werelabeled with 5 μM of Carboxy-Fluorescein-Succinimidyl Esther (CFSE). 10⁵CD4 T cells were then plated in a 96 well plate together with matureallo-DCs (5:1) in presence or absence of blocking anti-PD-1 antibodyaPD1(0376) (=PD1-0103-0312, as described herein before or in PCTApplication PCT/EP2016/073248) alone or in combination with chimericanti-LAG-3 antibodies (aLAG3(0403) to aLAG(0418)) or referenceantibodies (humanized BAP050 (LAG525) and BMS 986016) at theconcentration of 10 μg/ml. DP47 is a non-binding human IgG with a LALAmutation in the Fc portion to avoid recognition by FcγR and was used asnegative control.

Five days later the cell-culture supernatants were collected and used tomeasure the IL-2 levels by ELISA (R&D systems), and the cells were leftat 37° C. for additional 5 hours in presence of Golgi Plug (Brefeldin A)and Golgi Stop (Monensin). The cells were then washed, stained on thesurface with anti-human CD4 antibody and the LiveDead fixable dye Aqua(Invitrogen) before being fixed/permeabilized with Fix/Perm Buffer (BDBioscience). We performed intracellular staining for Granzyme B (BDBioscience) and IFN-γ (eBioscience). Results are shown in FIGS. 2A and2B.

Effect of PD-1 and LAG-3 Blockade on Cytotoxic Granzyme B Release byHuman CD4 T Cells Cocultured with a B Cell-Lymphoblatoid Cell Line(ARH77).

In functional studies, CD4 T cells were co-cultured with the tumor cellline ARH77, a B cell lymphoblastoid cell line which expresses lowerlevels of PDL-1 than mDCs, to better characterize the contribution ofLAG-3 antagonism to PD-1 blockade. The rest of the experimental set upand readout remained unchanged from the mMLR. The anti-LAG-3 antibodies(aLAG3(0414) and aLAG3(0416), chosen based on their ability toco-secrete IL-2 and Granzyme B in the mMLR) in combination withanti-PD-1 antibody caused a more significant increase in Granzyme Bsecretion by CD4 T cells than reference anti-LAG-3 antibodies((humanized BAP050 (LAG525) and BMS 986016)) (P<0.05) and anti-PD-1alone (P<0.01) as shown in FIG. 3.

Effect of PD-1 and LAG-3 Blockade on Treg Suppression of Granzyme B andIFN-γ Release by Human CD4 T Cells Cocultured with Irradiated AllogeneicPBMCs.

In functional studies involving regulatory T cells (Treg)-suppressionassays, PBMCs from the same donor where divided in two samples: one wasenriched in CD4 T cells and the other one in Tregs defined asCD4⁺CD25^(high) CD127^(low) T cells via a microbead kit (MiltenyiBiotec). Once purified the two populations, CD4 T cells were labelledwith 5 M of Carboxy-Fluorescein-Succinimidyl Esther (CFSE) while Tregswith 5 μM Cell-Trace-Violet (CTV) to be able to distinguish them at theFACS later on.

Both CD4 T cells (10⁵) and Tregs (10⁵) were then co-cultured in a 96well plate at 1:1 ratio together with irradiated. CD4-depleted PBMCs(10⁵) from an unrelated donor in presence or absence of anti-LAG-3antibodies (aLAG3(0414) and aLAG3(0416) or reference anti-LAG-3antibodies (humanized BAP050 (LAG525) and BMS 986016) in combinationwith anti-PD-1 antibody aPD1(0376) at the concentration of 10 μg/ml. Ascontrol to estimate the magnitude of the suppression of CD4 T celleffector functions by Tregs, CD4 T cells (10⁵) were also co-culturedwith irradiated PBMCs (10⁵) in the absence of Tregs.

Five days later the cell-culture supernatants were collected and usedlater to measure IFN-γ levels by ELISA (R&D systems), and the cells wereleft at 37° C. for additional 5 hours in presence of Golgi Plug(Brefeldin A) and Golgi Stop (Monensin). The cells were then washed,stained on the surface with anti-human CD4 antibody and the Live/Deadfixable dye Aqua (Invitrogen) before being fixed/permeabilized withFix/Perm Buffer (BD Bioscience). Intracellular staining was performedfor Granzyme B (BD Bioscience) and IFN-γ (eBioscience). Results areshown in FIGS. 4A and 4B.

The anti-LAG-3 antibodies (aLAG3(0414) and aLAG3(0416), in combinationwith anti-PD-1 antibody aPD1(0376) (=PD1-0103-0312, from PCT ApplicationPCT/EP2016/073248) elicited Tconv escape from regulatory T cell tightcontrol as demonstrated by the secretion of significantly higher amountof Granzyme B than Tconv in presence of anti-PD-1 alone (P<0.05) or inabsence of checkpoint inhibitors (P<0.001). Reference anti-LAG-3antibodies (humanized BAP050 (LAG525) and BMS 986016) in combinationwith anti-PD-1 did not significantly rescue Tconv effector functionsfrom Treg suppression. Similar results were obtained for IFN-γ even ifthe difference did not reach statistical significance with only 4donors.

Effect of PD-1 and LAG-3 Blockade on Granzyme B and IFN-γ Secretion byCD4 T Cells from Melanoma Patient PBMCs after Recall with ImmunogenicMelanoma-Antigen Peptide pools.

It has been previously described that melanoma patient PBMCs containdetectable frequencies of tumor-antigen specific T cells. Therefore, forPOC purposes, we tested anti-LAG-3 antibody (0414) plus anti-PD-1 versusor anti-PD-1 alone on melanoma patient PBMCs re-stimulated overnightwith immunogenic melanoma associated antigens peptide pools.

10⁵ to 10⁶ PBMCs from melanoma patients where incubated at roomtemperature in presence or absence of saturating concentrations (10μg/ml) of anti-PD-1 alone (0376), in combination with anti-LAG-3(aLAG3(0414)=(0414), 10 μg/ml) antibody. T cells were then re-stimulatedover-night with a pool of immunogenic tumor related antigens likeMAGEA1, MAGEA3, MAGEA4, Melan-A/MART-1, NYESO-1, Melanocyte protein Pmel17 gp100, Tyrosinase. Tyrosinase-related protein 2 in presence ofprotein transport inhibitors Golgi Plug (Brefeldin A) and Golgi Stop(Monensin).

The cells were then washed, stained on the surface with anti-human CD4antibody and the Live/Dead fixable dye Aqua (Invitrogen) before beingfixed/permeabilized with Fix/Perm Buffer (BD Bioscience). Intracellularstaining was performed for Granzyme B (BD Bioscience) and IFN-γ(eBioscience).

The combination of anti-LAG-3 and anti-PD-1 antibodies (P<0.01 andP<0.001) significantly (P<0.01 and P<0.0001) enhanced tumor-antigenspecific T cell effector functions (i.e. Granzyme B and IFN-γ secretion)while PD-1 blockade alone did not show any effect (data not shown).

Example 10 Generation and Production of Bispecific Anti-PD1/Anti-LAG3Antibodies

10.1 Production and Expression of Bispecific Antibodies which Bind toPD1 and LAG3 with VH/VL Domain Exchange/Replacement (CrossMAb^(Vh-VL))in One Binding Arm and with Single Charged Amino Acid Substitutions inthe CH1/CL Interface

Multispecific antibodies which bind to human PD1 and human LAG3 weregenerated is described in the general methods section by classicalmolecular biology techniques and were expressed transiently in 293F ofExpi293F cells as described above. The multispecific 1+1CrossMAb^(Vh-VL) antibodies are described also in WO 2009/080252. Themultispecific antibodies were expressed using expression plasmidscontaining the nucleic acids encoding the amino acid sequences depictedin Table 14. A schematic structure of the 1+1 CrossMAb^(Vh-VL)bispecific antibodies is shown in FIG. 1A.

TABLE 14 Amino acid sequences of light chains (LC) and heavy chains(HC), with VH/VL domain exchange/replacement (1 + 1 CrossMAb^(Vh-Vl))1 + 1 Antibody HC1 HC2 LC1 LC2 PD1/LAG3 SEQ ID NO: SEQ ID NO: SEQ ID NO:SEQ ID NO: 0799 PD1  96  97  98  99 (0376)/aLAG3 (0416) PD1/LAG3 SEQ IDNO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 0927 PD1  96 100  98 101(0376)/aLAG3 (0414) PD1/LAG3 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:0222 PD1 102 103 104 105 (0069)/aLAG3 (25F7) PD1/LAG3 SEQ ID NO: SEQ IDNO: SEQ ID NO: SEQ ID NO: 0224 PD1 106 103 107 105 (0098)/aLAG3 (25F7)

For all constructs knobs into holes heterodimerization technology wasused with a typical knob (T366W) substitution in the first CH3 domainand the corresponding hole substitutions (T366S, L368A and Y410V) in thesecond CH3 domain (as well as two additional introduced cysteineresidues S354C/Y349′C) (contained in the respective corresponding heavychain (HC) sequences depicted above).

10.2 Production and Expression of multispecific Antibodies which Bind toPD1 and LAG3 with CH1/Ck Domain Exchange/Replacement (2+2CrossMab^(CH1/Ck)) in Two Binding Arms and with Charged Amino AcidSubstitutions in the CH1/CL Interfaces of the Other

In this example multispecific antibodies which bind to human PD1 andhuman TIM3 were generated as described in the general methods section byclassical molecular biology techniques and were expressed transiently in293F of Expi293F cells as described above. The multispecific 2+2CrossMAb^(CH1/Ck) antibodies are described also in WO 2010/145792. Themultispecific antibodies were expressed using expression plasmidscontaining the nucleic acids encoding the amino acid sequences depictedin Table 15. A schematic structure of the 2+2 CrossMAb^(CH1/Ck)bispecific antibodies is shown in FIG. 1A.

TABLE 15 Amino acid sequences of light chains (LC) and heavy chains(HC), with VH/VL domain exchange/replacement (2 + 2 CrossMAb^(CH1/Ck))2 + 2 Antibody 2× HC 2× LC1 2× LC2 PD1/LAG3 SEQ ID NO: 114 SEQ ID NO:115 SEQ ID NO: 101 8970 PD1 (0376)/aLAG3 (0414) PD1/LAG3 SEQ ID NO: 116SEQ ID NO: 115 SEQ ID NO: 99  8984 PD1 (0376)/aLAG3 (0416) PD1/LAG3 SEQID NO: 117 SEQ ID NO: 115 SEQ ID NO: 105 9010 PD1 (0376)/aLAG3 (25F7)10.3 Production and Expression of Multispecific Antibodies which Bind toPD1 and LAG3 with CH1/Ck Domain Exchange/Replacement (2+1CrossMab^(CH1/Ck)) in One Binding Arm (PD1 crossFab Fused to theC-Terminus of the Fc Knob Heavy Chain) and with Charged Amino AcidSubstitutions in the CH1/CL Interfaces of the Other

In this example multispecific antibodies which bind to human PD1 andhuman TIM3 were generated as described in the general methods section byclassical molecular biology techniques and were expressed transiently in293F of Expi293F cells as described above. Multispecific 2+1CrossMAb^(CH1/Ck) antibodies are described also in WO2013/026831. Themultispecific antibodies were expressed using expression plasmidscontaining the nucleic acids encoding the amino acid sequences depictedin Table 16. A schematic structure of the 2+1 CrossMAb^(CH1/Ck)bispecific antibodies is shown in FIG. 1B.

TABLE 16 Amino acid sequences of light chains (LC) and heavy chains(HC), with CH1/Ck domain exchange/replacement (2 + 1 CrossMab^(CH1/Ck))2 + 1 Antibody HC1 HC2 LC1 2× LC2 PD1/LAG3 SEQ ID NO: SEQ ID NO: SEQ IDNO: SEQ ID NO: 8310 aLAG3 118 119 115 101 (0414)/PD1 (0376) PD1/LAG3 SEQID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: 8311 aLAG3 120 121 115  99(0416)/PD1 (0376) PD1/LAG3 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:1252 aLAG3 122 103 115 105 (25F7)/PD1 (0376)

Alternatively, the PD1 crossFab fused to the C-terminus of the Fc knobheavy chain can be replaced by single chain Fab (scFab). Suchmultispecific 2+1 antibodies comprising a scFab are described also inWO2010/136172 and can be expressed using expression plasmids containingthe nucleic acids encoding the amino acid sequences depicted in Table17. A schematic structure of the 2+1 bispecific antibodies with a scFabfused at the C-terminus of the Fc knob heavy chain is shown in FIG. 1C.

TABLE 17 Amino acid sequences of light chains (LC) and heavy chains(HC), with PD1 scFab 2 + 1 Antibody HC1 HC2 2× LC PD1/LAG3 8312 SEQ IDNO: SEQ ID NO: SEQ ID NO: aLAG3(0414)/ 123 119 101 PD1(0376) PD1/LAG38313 SEQ ID NO: SEQ ID NO: SEQ ID NO: aLAG3(0416)/ 124 121  99 PD1(0376)PD1/LAG3 1088 SEQ ID NO: SEQ ID NO: SEQ ID NO: aLAG3(25F7)/ 125 103 105PD1(0376)10.4 Production and Expression of Multispecific Antibodies which Bind toPD1 and LAG3 with VH/VL Fused Each at a C-Terminus of the Heavy Chains(2+1 PRIT Format)

In this example multispecific antibodies which bind to human PD1 andhuman TIM3 were generated as described in the general methods section byclassical molecular biology techniques and were expressed transiently in293F of Expi293F cells as described above. This type of multispecific2+1 antibodies is also described in WO 2010/115589. The multispecificantibodies were expressed using expression plasmids containing thenucleic acids encoding the amino acid sequences depicted in Table 18. Aschematic structure of the 2+1 PRIT-type bispecific antibodies is shownin FIG. 1D.

TABLE 18 Amino acid sequences of light chains (LC) and heavy chains(HC), with VH and VL domain fused C-terminally to heavy chains 2 + 1Antibody HC1 HC2 2× LC PD1/LAG3 0918 SEQ ID NO: SEQ ID NO: SEQ ID NO:aLAG3(25F7)/ 126 127 109 aPD1(0376)10.5 Production and Expression of multispecific Antibodies which Bind toPD1 and LAG3 with VH/VL Domain Exchange/Replacement (1+1CrossMab^(VH/VL) Trans Format) in One Binding Arm and with Charged AminoAcid Substitutions in the CH1/CL Interfaces of the LAG3 Fab Fused to theC-Terminus of the Fc Hole Heavy Chain

Multispecific antibodies which monovalently bind to both human PD1 andto human LAG3 were produced wherein a LAG3 Fab is fused via its variableheavy domain to the C-terminus of one of the heavy chains, preferablythe Fc hole heavy chain. The molecules were generated as described inthe general methods section by classical molecular biology techniquesand were expressed transiently in 293F of Expi293F cells as describedabove. The multispecific antibodies were expressed using expressionplasmids containing the nucleic acids encoding the amino acid sequencesdepicted in Table 19. A schematic structure of the 1+1 CrossMab^(VH/VL)trans-type bispecific antibodies is shown in FIG. 1H.

TABLE 19 Amino acid sequences of light chains (LC) and heavy chains(HC), with aLAG3 Fab fused C-terminally to heavy chains 1 + 1 AntibodyHC1 HC2 LC1 LC2 PD1/LAG3 SEQ ID SEQ ID SEQ ID SEQ ID 0725 aLAG3 NO: 96NO: 144 NO: 98 NO: 101 (0414)/aPD1 (0376)10.6 Production and Expression of Multispecific Antibodies which Bind toPD1 and LAG3 with VH/VL Domain Exchange/Replacement (2+1CrossMab^(VH/VL) Trans Format) in One Binding Arm and with Charged AminoAcid Substitutions in the CH1/CL Interfaces of the Two LAG3 Fabs, One ofthem Fused to the C-Terminus of the Fc Hole Heavy Chain

Multispecific antibodies which monovalently bind to human PD1 andbivalently bind to human LAG3 were produced wherein a LAG3 Fab is fusedvia its variable heavy domain to the C-terminus of one of the heavychains, preferably the Fc hole heavy chain. The molecules were generatedas described in the general methods section by classical molecularbiology techniques and were expressed transiently in 293F of Expi293Fcells as described above. The multispecific antibodies were expressedusing expression plasmids containing the nucleic acids encoding theamino acid sequences depicted in Table 20. A schematic structure of the2+1 CrossMab^(VH/VL) trans-type bispecific antibodies is shown in FIG.1I.

TABLE 20 Amino acid sequences of light chains (LC) and heavy chains(HC), wherein one of the aLAG3 Fabs is fused C-terminally to heavychains 1 + 1 Antibody HC1 HC2 LC1 2× LC2 PD1/LAG3 SEQ ID NO: SEQ ID NO:SEQ ID NO: SEQ ID NO: 0750 aLAG3 96 145 98 101 (0414)/aPD1 (0376)10.7 Purification and Characterization of Multispecific Antibodies whichBind to PD1 and TIM3

The multispecific antibodies expressed above were purified from thesupernatant by a combination of Protein A affinity chromatography andsize exclusion chromatography. All multispecific antibodies can beproduced in good yields and are stable. The obtained products werecharacterized for identity by mass spectrometry and analyticalproperties such as purity by SDS-PAGE, monomer content and stability

Mass Spectrometry

The expected primary structures were analyzed by electrospray ionizationmass spectrometry (ESI-MS) of the deglycosylated intact CrossMabs anddeglycosylated/plasmin digested or alternatively deglycosylated-limitedLysC digested CrossMabs.

The CH1/Ck CrossMabs were deglycosylated with N-Glycosidase F in aphosphate or Tris buffer at 37° C. for up to 17 h at a proteinconcentration of 1 mg/ml. The plasmin or limited LysC (Roche) digestionswere performed with 100 μg deglycosylated CH1/Ck CrossMabs in a Trisbuffer pH 8 at room temperature for 120 hours and at 37° C. for 40 min,respectively. Prior to mass spectrometry the samples were desalted viaHPLC on a Sephadex G25 column (GE Healthcare). The total mass wasdetermined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik)equipped with a TriVersa NanoMate source (Advion).

Stability of Multispecific Antibodies

In order to assess stability of the antibody constructs, thermalstability as well as aggregation onset temperatures are assessedaccording to the following procedure. Samples of the indicatedantibodies are prepared at a concentration of 1 mg/mL in 20 mMHistidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into a 10μL micro-cuvette array and static light scattering data as well asfluorescence data upon excitation with a 266 nm laser are recorded withan Optim1000 instrument (Avacta Inc.), while the samples are heated at arate of 0.1° C./min from 25° C. to 90° C.

The aggregation onset temperature (T_(agg)) is defined as thetemperature at which the scattered light intensity starts to increase.The melting temperature (T_(m)) is defined as the inflection point in afluorescence intensity vs. wavelength graph.

Example 11 Characterization of Bispecific Anti-PD1/Anti-LAG3 Antibodies11.1 Binding ELISA ELISA for hu PD1

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated PD1-ECD-AviHis at a concentration of500 ng/ml and incubated at 4° C. over night. After washing (3×90 μl/wellwith PBST-buffer) 25 μl anti PD1 antibody samples were added inincreasing concentrations and incubated 1 h at RT. After washing (3×90μl/well with PBST-buffer) 25 μl/well goat-anti-human H+L-POD (JIR,JIR109-036-098) was added in 1:5000 dilution and incubated at RT for 1 hon a shaker. After washing (3×90 μl/well with PBST-buffer) 25 μl/well ofTMB substrate (Roche, 11835033001) was added and incubated until OD 2-3.Measurement took place at 370/492 nm.

Cell ELISA for Human PD1

Adherent CHO-K1 cell line stably transfected with plasmid15311_hPD1-fl_pUC_Neo coding for full-length human PD1 and selectionwith G418 (Neomycin restistance marker on plasmid) were seeded at aconcentration of 0.01×10E6 cells/well in 384-well flat bottom plates andgrown over night.

The next day 25 μl/well PD1 sample or human anti PD1 (Roche)/mouse antiPD1 (Biolegend; cat.:329912) reference antibody were added and incubatedfor 2 h at 4° C. (to avoid internalization). After washing carefully(1×90 μl/well PBST) cells were fixed by adding 30 μl/well 0.05%Glutaraldehyde (Sigma, Cat.No: G5882, 25%) diluted in 1×PBS-buffer andincubated for 10 min at RT. After washing (3×90 μl/well PBST) 25 μl/wellsecondary antibody was added for detection: goat-anti-human H+L-POD(JIR, JIR109-036-088)/Sheep-anti-mouse-POD (GE NA9310) followed by 1 hincubation at RT on shaker. After washing (3×90 μl/well PBST) 25 μl/wellTMB substrate solution (Roche 11835033001) was added and incubated untilOD 1.0-2.0. Plates were measured at 370/492 nm.

Cell ELISA results are listed as “EC₅₀ CHO-PD1”-values [nM] in Table 21below.

ELISA for Human Lag3

Nunc maxisorp plates (Nunc 464718) were coated with 25 μl/wellrecombinant Human LAG-3 Fc Chimera Protein (R&D Systems, 2319-L3) at aprotein concentration of 800 ng/ml and incubated at 4° C. overnight orfor 1 h at room temperature. After washing (3×90 μl/well withPBST-buffer) each well was incubated with 90 μl blocking buffer (PBS+2%BSA+0.05% Tween 20) for 1 h at room temperature. After washing (3×90μl/well with PBST-buffer) 25 μl anti-Lag3 samples at a concentration of1-9 μg/ml (1:3 dilutions in OSEP buffer) were added and incubated 1 h atRT. After washing (3×90 μl/well with PBST-buffer) 25 μl/well goatanti-Human Ig κ chain antibody-HRP conjugate (Milipore, AP502P) wasadded in a 1:2000 dilution and incubated at RT for 1 h. After washing(3×90 μl/well with PBST-buffer) 25 μl/well TMB substrate (Roche,11835033001) was added and incubated for 2-10 min. Measurement tookplace on a Tecan Safire 2 instrument at 370/492 nm.

Cell-Surface Lag3 Binding ELISA

25 μl/well of Lag3 cells (recombinant CHO cells expressing Lag3, 10000cells/well) were seeded into tissue culture treated 384-well plates(Corning, 3701) and incubated at 37° C. for one or two days. The nextday after removal of medium, 25 μl anti-Lag3 samples (1:3 dilutions inOSEP buffer, starting at a concentration of 6-40 nM) were added andincubated for 2 h at 4° C. After washing (1×90 μl in PBST) cells werefixed by addition of 30 μl/well glutaraldehyde to a final concentrationof 0.05% (Sigma Cat.No: G5882), 10 min at room temperature. Afterwashing (3×90 μL/well with PBST-buffer) 25 μl/well goat anti-Human Ig κchain antibody-HRP conjugate (Milipore, AP502P) was added in a 1:1000dilution and incubated at RT for 1 h. After washing (3×90 μl/well withPBST-buffer) 25 μl/well TMB substrate (Roche, 11835033001) was added andincubated for 6-10 min. Measurement took place on a Tecan Safire 2instrument at 370/492 nm. Cell ELISA results are listed as “EC₅₀CHO-LAG3”-values [nM] in Table 21 below.

Inhibition of LAG-3 Binding to MHC-II Expressed on Human A375 TumorCells (by ELISA)

25 μl/well of A375 cells (10000 cells/well) were seeded into tissueculture treated 384-well plates (Corning, 3701) and incubated at 37° C.overnight. Anti-Lag3 antibodies were pre-incubated for 1 h withbiotinylated-Lag3 (250 ng/ml) in cell culture medium in 1:3 dilutionsstarting at 3 μg/ml antibody-concentration. After removal of medium fromthe wells with the seeded cells, 25 μl of the antibody-Lag3pre-incubated mixtures were transferred to the wells and incubated for 2h at 4° C. After washing (1×90 μl in PBST) cells were fixed by additionof 30 μl/well glutaraldehyde to a final concentration of 0.05% (SigmaCat.No: G5882), 10 min at room temperature. After washing (3×90 μl/wellwith PBST-buffer) 25 μl/well Poly-HRP40-Streptavidin (Fitzgerald,65R-S104PHRPx) was added in a 1:2000 or 1:8000 dilution and incubated atRT for 1 h. After washing (3×90 μl/well with PBST-buffer) 25 μl/well TMBsubstrate (Roche, 11835033001) was added and incubated for 2 to 10 min.Measurement took place on a Tecan Safire 2 instrument at 370/492 nm.Inhibition ELISA results are listed as “IC₅₀ MHCII/ELISA”-values [nM] inTable 21 below.

TABLE 21 Summary of Binding of different bispecific anti-PD1/anti-LAG3antibodies ELISA ELISA EC₅₀ EC₅₀ CHO- MHCII/ huPD1 huLAG3 CHO-PD1 LAG3ELISA Bispecific rel. EC₅₀ rel. EC₅₀ rel. EC₅₀ rel. EC₅₀ IC₅₀ antibody[nM] [nM] [nM] [nM] [nM] PD1/LAG3 0927 0.07 0.18 0.1 0.23 1.11(PD1-0376/LAG3- 0414) (1 + 1) PD1/LAG3 0799 0.06 0.07 0.07 0.20 0.72(PD1-0376/LAG3- 0416) (1 + 1) PD1/LAG3 0222 0.16 1.14 0.28 0.72 0.77(PD1-0069/LAG3 25F7) (1 + 1) PD1/LAG3 0224 0.04 0.86 0.06 0.86 0.79(PD1-0098/LAG3 25F7) (1 + 1) PD1/LAG3 8310 0.06 0.06 0.34 0.20 0.47(PD1-0376/LAG3- 0414) (1 + 2) PD1/LAG3 8311 0.05 0.06 0.32 0.17 0.39(PD1-0376/LAG3- 0416) (1 + 2) PD1/LAG3 1252 0.03 0.02 0.31 0.64 0.47(PD1-0376/LAG3 25F7) (1 + 1) PD1/LAG3 8970 0.05 0.04 0.46 0.20 0.45(PD1-0376/LAG3- 0414) (2 + 2) PD1/LAG3 8984 0.05 0.05 0.54 0.17 0.44(PD1-0376/LAG3- 0416) (2 + 2) PD1/LAG3 9010 0.04 0.05 0.36 0.48 0.52(PD1-0376/LAG3- 25F7) (2 + 2)

11.2 Binding Biacore

Antigen Binding Properties of Multispecific Antibodies which Bind to PD1and LAG3

Binding of the multispecific antibodies to their respective targetantigens, i.e. PD1 and TIM3, was assessed by Biacore).

PD1 Binding can be Assessed According to the Following Procedure:

Anti-human Fc IgG was immobilized by amine coupling to the surface of a(Biacore) CM5 sensor chip. The samples were then captured and hu PD1-ECDwas bound to them. The sensor chip surface was regenerated after eachanalysis cycle. The equilibrium constant and kinetic rate constants werefinally gained by fitting the data to a 1:1 Langmuir interaction model.

About 10,000 response units (RU) of 20 μg/ml anti-human IgG (GEHealthcare #BR-1008-39) were coupled onto all flow cells of a CM5 sensorchip in a Biacore T200 using an amine coupling kit supplied by GEHealthcare. The sample and running buffer was HBS-EP+(0.01 M HEPES, 0.15M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20, pH 7.4). Flow celltemperature was set to 25° C. and sample compartment temperature to 12°C. The system was primed with running buffer.

Different samples were injected for 15 seconds with a concentration of10 nM and consecutively bound to the flow cells 2, 3 and 4. Then acomplete set of human PD1-ECD concentrations (300 nM, 100 nM, 2×33.3 nM,11.1 nM, 3.7 nM, 1.2 nM and 2×0 nM) was injected over each sample for300 s followed by a dissociation time of 10/600 s and two 30 sregeneration steps with 3 M MgCl₂, of which the last one contained an“extra wash after injection” with running buffer. Finally the doublereferenced data was fitted to a 1:1 Langmuir interaction model with theBiacore T200 Evaluation Software.

LAG3 Binding was Assessed According to the Following Procedure:

A Biacore SA CAP Kit provided by GE Healthcare was used to perform thisassay. The kinetic values were obtained at 25° C. in HBS-EP+ (GeHealthcare) buffer.

The SA CAP Chip was docked to a Biacore T200 as prescribed in the manualof the CAP Kit. The run method contains four commands. Firstly CAPreagent was injected for 300 s at a flow rate of 10 μl/min to hybridizethe immobilized single stranded DNAs using a ‘General’ command. Thecommand is followed by a 15 seconds long injection of a 1 μg/ml dilutionof biotinylated Fc-tagged, human Lag3 extra cellular domain in runningbuffer. This results in a capture level of about 50 RU. A single-cyclecommand was used to inject five different sample concentrations (100nM-6.25 nM, 2-fold dilutions) followed by a 1200 seconds longdissociation phase. The chip was then regenerated as prescribed in theSA CAP Kit manual.

Finally, the obtained curves were evaluated using the Biacore T200Evaluation software version 3.0.

Results:

The interactions did not fit to a 1:1 Langmuir binding model, becauseall samples aside from 0799 and 0927 have two Lag3 binding moieties andtherefore show avidity. Since 0799 and 0927 contained a smallmiss-paired, bivalent sample population, they also showed some avidity.

Therefore the sensorgrams were only ranked according to their off rates.This was done by visual comparison of the single-cycle kinetic curves.By doing this, it was shown that the 0416-Lag3 ECD complex is morestable than any other in this sample set. The monovalent <Lag3> Crossmabformat (0799) still displays a slower offrate than any other sample inthis experiment.

Furthermore, it was seen that the affine Lag3-0414/Lag3-0927-Lag3 ECDcomplex is the weakest of those, observed in this experiment. The affinebinding portions of 0414 and 0416 are roughly comparable to the affineportions of their monovalent Crossmab counter parts 0927 and 0799.Results are indicated in Table 22.

TABLE 22 Binding Quality of PD1-LAG3 Bispecific Antibodies determined bySPR measurement Sample Binding quality aLAG3(0414) ++ aLAG3(0416) +++PD1/LAG3 0927 (1 + 1) + PD1/LAG3 0799 (1 + 1) +++ aLAG3(25F7) ++aLAG3(MDX26H10) ++ aLAG3(BMS986016) ++ aLAG3(BAP050) +

Avidity Assessment of the Trans Formats Compared to the 1+1 BispecificAntibodies 0927 and 0799:

The dissociation constants of the bispecific molecules (sample) andtheir individual targets as well as a combination of PD1 and LAG3(analyte) were determined to assess the avidity gain provided by bindingwith all valences at the same time.

Previous to the measurement on a Biacore 8K, a CM5 sensor chip wasprepared using the standard amine coupling kit provided by GEHealthcare. An in-house produced antibody directed against a specificmutation in the Fc part of the sample (i.e. an Fc part carrying thePGLALA mutations), herein called anti-PGLALA antibody (such antibodiesare described in WO 2017/072210), was therefore diluted to aconcentration of 50 μg/ml in acetate buffer pH 5.0. It was coupled toall flow cells and channels at a 8 μl/min flow speed over 1200 s,yielding in a bound response of about 19000 RU.

HBS-EP⁺ buffer (GE HC) was used as running buffer for the sensor chippreparation as well as the main run itself. The analysis started after astartup consisting of three 17 s long sample injections followed by aregeneration step utilizing a 10 mM NaOH solution. In a first step, thedifferent samples were captured by the anti-PGLALA antibody onto theindividual channels' flow-cell two on the sensor chip surface byinjecting it for 17 s at a flow rate of 10 μL/min. Secondly, one of thethree analytes (PD1, LAG3-Fc, 2+2 PD1/LAG3-Fc Fusion) was injected intoboth flow-cells for 200 s at a flow rate of 50 μl/min followed by a 1000s long (600 s in case of the LAG3-Fc) dissociation phase. Finally, theanti-PGLALA antibody/sample complex was dissolved by two consecutiveinjections (30 s long) of 10 mM NaOH. Each individual kineticdetermination consisted of four cycles with different analyteconcentrations (0 nM, 5 nM, 25 nM and 100 nM).

The resulting data was evaluated using the Biacore 8K EvaluationSoftware. A 1:1 dissociation fit was applied and resulting kd valueswere converted into complex half-life in minutes. The difference betweenthe avidity binding of the PD1/Lag3-Fc fusion antigen binding moleculeand its main individual contributor (either PD1 or Lag3) was calculatedand sorted into one of three categories describing the stability gain bymultivalent and bispecific binding (Table 23).

TABLE 23 Increase of Complex Stability provided by the avidity ofPD1-LAG3 Bispecific Antibodies determined by SPR measurement SampleBinding quality PD1/LAG3 0927 (1 + 1) ++ PD1/LAG3 0799 (1 + 1) +++PD1/LAG3 0725 (1 + 1 trans) + PD1/LAG3 0750 (1 + 2 trans) ++11.3 Dimerization of Cellular PD1 and LAG3 after Simultaneous EngagementVia Bispecific Anti-PD1/Anti-LAG3 Bispecific Antibodies

Bispecific anti-PD1/anti-LAG3 antibodies were generated in variousformats as described in Example 10. This cellular assay was used todemonstrate the dimerization or at last binding/interaction of twodifferent receptors, which are cytosolically fused with two fragments ofan enzyme, upon ligation or cross-linking with a bispecific antibodyagainst both targets. Hereby only one receptor alone shows no enzymaticactivity. For this specific interaction, the cytosolic C-terminal endsof both receptors were individually fused to heterologous subunits of areporter enzym. A single enzyme subunit alone showed no reporteractivity. However, simultaneous binding of an anti-PD1/anti-LAG3bispecific antibody construct to both receptors was expected to lead tolocal cytocolic accumulation of both receptors, complementation of thetwo heterologous enzyme subunits, and finally to result in the formationof a specific and functional enzyme that hydrolyzes a substrate therebygenerating a chemiluminescent signal.

In order to analyze the cross-linking effect of the bispecificanti-PD1/anti-LAG3 antibodies, 10,000 PD1⁺ LAG3⁺ human U2OS cells/wellwere seeded into white flat bottom 96-well plates (costar, cat.no.#3917) and cultured overnight in assay medium. On the next day cellmedium was discarded and replaced by fresh medium. Antibody or liganddilutions were prepared and titrated amounts of indicated (bispecific)antibodies were added and incubated at 37° C. for 2 hours. Next, asubstrate/buffer mix (e.g. PathHunterFlash detection reagent) was addedand again incubated for 1 h. For measuring chemoluminescence inducedupon simultaneous binding and dimerization a Tecan infinite reader wasused.

The results are shown in FIGS. 5A and 5B. Plotted is thechemoluminescence (measured in RU) against the antibody concentration.Monospecific (bivalent) anti-LAG3 antibodies were not able to provoke achemoluminescence signal whereas all bispecific anti-PD1/anti-LAG3antibodies induced a chemoluminescence signal in a concentrationdependent manner.

To show the specificity of the simultaneous binding (and induction of aluminiscence signal) a competition experiment was performed: As shownbefore treatment with a bispecific antibody (1252) induced aluminiscence signal in a dose-dependent fashion (FIG. 5C). If the samebispecific antibody was provided in the presence of either an aLAG3antibody (0156. MDX25F7) or anti-PD1 antibody (0376), the signal waseither almost inhibited (for PD1 competition) or at least significantlyreduced (LAG3). Both parental antibodies are the same binders ascomprised in the bispecific antibody (1252, 2+1 LAG3/PD1-format). Thecompeting antibodies were given each at a constant concentration of 20μg/ml.

The results of a further experiment are shown in FIG. 5D. Similar to theprevious competition experiment the incubation with parental aLAG3(0156) or PD1 antibodies (0376, each constantly at 101 g/ml) had aneffect on the binding properties of the bispecific antibody (1252, 2+1format of the bispecific aLAG3-0156 and PD11-0376) to PD1 Lag3double-expressing cells, as measured by the luminiscence signal.Competition with anti-PD1 antibody (0376) and also recombinant LAG3:Fcprotein (0160) almost abolished the signal, whereas presence of thesingle aLAG3 binder (0156) only led to partial inhibition. The twofurther anti-LAG3 antibodies 0414 and 0416, which are binding to adifferent epitope than 0156, did not compete for binding with thebispecific antibody comprising aLAG3 binder (0156), because they did notmodulate the signal significantly.

In a further experiment, the simultaneous binding of bispecificanti-LAG3/anti-PD1 antibodies comprising different aLAG3 binder (0414vs. 0416) and different formats (1+1 vs. 2+1) was compared (FIGS. 6A to6D). As described before, several anti-LAG3/anti-PD1 bispecificantibodies were tested, either in an 1+1 CrossMab format (0799 and 0927)or 2+1 format (two Lag3 binding arms and one PD1 crossFab fragment fusedC-terminal: 8311 and 8310). In FIGS. 6A and 6B the curves (absorbancevs. concentration) for the constructs with binder aLAG3-0416 and inFIGS. 6C and 6D those for the corresponding constructs with aLAG4-0414are shown. All constructs tested were able to bind to the cells and toinduce chemoluminescence. The calculated EC₅ values for the bindingcurves are shown in Table 24 below.

TABLE 24 EC₅₀ values as measured in the dimerization binding assayBispecific Antibody Format MW [kD] EC₅₀ [pM] 0927 (PD1-0376/LAG3-0414)1 + 1 145 41 0799 (PD1-0376/LAG3-0416) 1 + 1 145 76 8310(PD1-0376/LAG3-0414) 1 + 2 193 28 8311 (PD1-0376/LAG3-0416) 1 + 2 193119

In another experiment, the simultaneous binding of bispecificanti-LAG3/anti-PD1 antibodies comprising different aLAG3 binder (0414vs. 0416) and different formats (2+1 vs. 2+2) was compared (FIGS. 7A to7D). Anti-LAG3/anti-PD1 bispecific antibodies were tested, either in or2+1 format (two LAG3 binding arms and one PD1 crossFab fragment fusedC-terminal: 8311 and 8310) or in 2+2 crossmab format (two LAG3 bindingarms and two PD1 crossFab fragments fused C-terminal: 8970 and 8984). InFIGS. 7A and 7B the curves (absorbance vs. concentration) for theconstructs with binder aLAG3-0414 and in FIGS. 7C and 7D those for thecorresponding constructs with aLAG4-0416 are shown. All constructstested were able to bind to the cells and to induce chemoluminescence.The calculated EC₅₀ values for the binding curves are shown in Table 25below.

TABLE 25 EC₅₀ values as measured in the dimerization binding assayBispecific Antibody Format MW [kD] EC₅₀ [pM] 8310 (PD1-0376/LAG3-0414)2 + 1 193 114 8311 (PD1-0376/LAG3-0416) 2 + 1 193 124 8970(PD1-376/LAG3-0414)  2 + 2 242 83 8984 (PD1-0376/LAG3-0416) 2 + 2 242 91

In a further experiment, the simultaneous binding of bispecificanti-LAG3/anti-PD1 antibody PD1/LAG3 0927 in the classical 1+1CrossMAb^(Vh-VL) format was compared with the bispecificanti-LAG3/anti-PD1 antibodies in the 1+1 trans format (PD1/LAG3 0725)and 2+1 trans format (PD1/LAG3 0750) For this experiment, the followingchanges to the method were applied. In order to analyze thecross-linking effect of the different anti-LAG3/anti-PD1 antibodyformats, 7500 PD1⁺ LAG3⁺ human U2OS cells/well were seeded into whiteflat bottom 96-well plates together with non-serial dilutions ofantibodies (final concentration of 0.29 pM to 5484 pM) and wereincubated for 20 h at 37° C. in a CO₂ incubator. Next, assay plates wereequilibrated to room temperature and a substrate/buffer mix(PathHunterFlash detection reagent, Discoverx) was added and againincubated for 4 h. For measuring chemoluminescence induced uponsimultaneous binding and dimerization a SpectraMax L plate reader(Molecular Devices) was used.

In FIG. 7E the dose-response curves (luminescence vs. concentration) ofthe PD1-LAG3 bispecific antibodies 1+1 CrossMab (0927), 1+1 transCrossMab with N-terminal aPD1 and C-terminal aLAG3 (0725) as well as 2+1trans CrossMab with N-terminal aPD1 and N-plus C-terminal aLAG3 (0750)are depicted. Compared to PD1/LAG3 0927 the PD1 LAG3 receptorcrosslinking effect of PD1/LAG3 0725 is clearly higher whereas it isslightly lower for PD1/LAG3 0750.

11.4 Measurement of Bispecific Anti-LAG3/Anti-PD1 Trans CrossMabVariants in a PD-1 & LAG-3 Combo Reporter Assay

To test the neutralizing potency of the different anti-PD1-LAG3 antibodyformats in restoring a suppressed T cell response in vitro, acommercially available reporter system was used. The PD1 & LAG3 combobioassay consists of PD1-, LAG3- and T cell receptor (TCR)-expressingreporter cells, MHC-II- and PDL1-expressing tumor cells and aTCR-activating-antigen.

The effector cells are Jurkat T cells expressing human PD1, human LAG3,a human TCR and a luciferase reporter driven by an NFAT response element(NFAT-RE). The target cells are A375 cells expressing human PD-L1. Inbrief, the reporter system is based on three steps: (1) TCRactivating-antigen-induced NFAT cell activation. (2) inhibition of theactivating signal mediated by the interaction between MHCII (A375 cells)and LAG3⁺ (Jurkat cells) as well as PD-L1 (A375 cells) and PD1 (Jurkatcells), and (3) recovery of the NFAT activation signal by PD1 andLAG3-antagonistic/neutralizing antibodies.

For this experiment, 1×10⁴ A375 target cells per well were incubatedovernight with TCR activating antigen (Promega) in 96-well flat bottomassay plates in a CO₂ incubator at 37° C. Next, media from plates wasremoved and serial dilutions (final assay concentration of 0.01 nM to857 nM) of anti-LAG3/anti-PD1 antibodies as well as 5×10⁴ Jurkateffector cells per well were added. After 6 hours of incubation at 37°C. in a CO₂ incubator, assay plates were equilibrated to roomtemperature and 80 μl ONE-Glo Ex substrate (Promega) was added to eachwell. After 10 min of incubation luminescence was measured in aSpectraMax L plate reader (Molecular Devices). Simultaneous binding ofan anti-PD1/anti-LAG3 bispecific antibody construct to both receptorswas expected to lead to local cytocolic accumulation of both receptors,complementation of the two heterologous enzyme subunits, and finally toresult in the formation of a specific and functional enzyme thathydrolyzes a substrate thereby generating a chemiluminescent signal. InFIG. 7F the dose-response curves (luminescence vs. concentration) of theanti-PD1/anti-LAG3 bispecific antibodies 1+1 CrossMab (0927), 1+1 transCrossMab with N-terminal aPD1 and C-terminal aLAG3 (0725) as well as 2+1trans CrossMab with N-terminal aPD1 and N-plus C-terminal aLAG3 (0750)are depicted. The ability of 0927 and 0725 to recover reporter cellactivation by blocking PD1 and LAG3 interaction with their respectiveligands is comparable whereas it is higher for 0750. This is furthermoreindicated by the EC₅₀ values listed in Table 26.

TABLE 26 EC₅₀ values as measured in the PD-1 & LAG-3 combo reporterassay Bispecific Antibody Format MW [kD] EC₅₀ [nM] PD1/LAG3 0927 1 + 1cis 145259 3.1 PD1/LAG3 0725 1 + 1 trans 145890 2.2 PD1/LAG3 0750 2 + 1trans 192888 0.6

Example 12 Functional Characterization of Bispecific Anti-PD1/Anti-LAG3Antibodies 12.1 Reduced Internalization Upon Binding to T-Cell SurfaceMeasurement of Receptor Internalization by Flow Cytometry

Receptor internalization represents an important sink for the moleculewhich can be degraded within few hours while the targeted receptors arerapidly re-expressed on the cell-surface ready to inhibitTCR-signalling. We therefore assessed receptor internalization upon thebinding of our constructs by flow cytometry where samples stained withdifferent bispecific formats at 4° C. were used as reference forcomparison with samples incubated at 37° C. for 3 hours after thestaining at 4° C.

Three days polyclonally activated CD4 T cells, previously cultured with1 mg/ml of plate bound anti-CD3 and 1 mg/ml of soluble anti-CD28antibodies, were incubated in presence of either anti-LAG3 oranti-PD1/anti-LAG3 bispecific antibodies (in duplicates) for 30 minutesat 4° C. The cells were then washed, divided in two groups, one of whichincubated for 3 additional hours at 37° C. and the other one wasimmediately stained with a labelled secondary antibody (eBioscience)before being fixed with BD Cell Fix. After the 3 hours incubations alsothe second group of the cells were stained with the labelled secondaryantibody before fixation. After staining, cells were washed two timeswith PBS/2% FCS and analyzed.

Therefore, the cells were acquired at LSRFortessa (BD Biosciences) andthe expression levels of detectable antibody on the cell surface werecompared among the two groups. The results are shown in FIG. 8B. Weobserved that after 3 hours all the bispecific formats as well as themonospecific bivalent aLAG-3 antibody have been internalized, howeverthe bispecific anti-PD1/anti-LAG3 antibodies in the 1+1 format (PD1/LAG30799 and PD1/LAG3 0927) were the least internalized.

Visualization of Antibody Localization and Internalization byFluorescence Confocal Microscopy

Activated CD4-positive cells were stained with CMFDA (Invitrogen) andplated on round coverslips treated with Retronectin (Takara Bio). Cellswere allowed 4 hours to adhere at 37° C. before fluorescently-taggedantibodies (1 μg/mL: a-LAG3 (1256), 1+1 PD1/LAG3 Bispec (0927), PD1-LAG31+2 Bispec (8310) and PD1-LAG3 2+2 Bispec (8970) labeled with Alexa 647)were added directly into growth media for different durations (15 min, 1hour and 3 hours). Cold PBS (Lonza) was used to quench the reaction andto wash off unbounded antibodies. Cells were then fixed with Cytofix(BD) for 20 minutes at 4° C. and washed twice with PBS (Lonza).Coverslips were then transferred and mounted on glass slides withFluoromount G (eBioscience) and kept in the dark at 4° C. overnightbefore imaging. A) The fluorescent images are shown in FIG. 9A. Thewhite signal represents the localization of the labeled antibody. B) Theintensity of the fluorescent signal from the membrane ROI, of highlytargeted cells, was divided by the intensity of the fluorescent signalfrom the cytoplasm ROI of the same cells, resulting in a ratio displayedin the Box Charts. In order to compare samples, One Way ANOVA analysisUncorrected Fisher's LSD was used (*=p<0.05; **=p<0.01). The results areshown in FIG. 9B. The analysis over time shows higher membranelocalization in the bispecific antibodies and LAG3 antibodies whencompared to intracellular clustering of TIM3 antibodies (used ascontrol). We observed that after 3 hours all the bispecific formats aswell as the monospecific bivalent aLAG-3 antibody have been internalizedwith the only exception of the 1+1 PD1/LAG3 Bispec (0927) (FIG. 9A).

Fluorescence confocal microscopy was performed with an inverted LSM 700from Zeiss with a 60× oil objective. Images were collected using Zensoftware (Zeiss) coupled to the microscope. The analysis of the imageswas performed with Imaris Software (Bitplane; Oxford Instrument) and thestatistical analysis was performed by GraphPad Prism (GraphpadSoftware).

12.2 Binding to Conventional T Cells Versus Tregs

A desired property of the lead PD1-LAG3 BsAb is the ability topreferentially bind to conventional T cells rather than to Tregs,because LAG3 on Tregs appears to negatively regulate their suppressivefunction. Therefore targeting LAG3 on Tregs with blocking antibodiescould be detrimental by increasing their suppressive function andeventually mask the positive blocking effect on other T cells. Wetherefore assessed the competitive binding of the differentanti-PD1/anti-LAG3 bispecific antibody formats to activated conventionaland regulatory T cells cultured together.

Regulatory T cells (Tregs) and conventional T cells (Tconv) were sortedfrom heathy donor PBMCs (Miltenyi), labelled with 5 mM CellTraceVioletor CFSE membrane dyes respectively and cultured together at 1:1 ratiofor 3 days with 1 mg/ml of plate bound anti-CD3 and 1 mg/ml of solubleanti-CD28 antibodies. On day 3 the cells were incubated for 30 min at 4°C. with either directly labelled anti-PD1, anti-LAG3 or bispecificantibodies, fixed with BD Cell Fix, and acquired at LSRFortessa (BDBiosciences).

While the monospecific anti-LAG3 parental antibody binds equally well toTregs and conventional T cells (FIG. 10A), the anti-PD1 counterpartbinds preferentially to conventional T cells due to higher expressionlevels of PD1 on effector T cells than on Tregs (FIG. 10B).Interestingly, also the 1+1 format of the PD1/LAG3 bispecific antibody(0927) retained the ability to preferentially bind to conventional Tcells than Tregs (FIG. 10C). This preferential binding to conventional Tcells can also be visualized by depicting the difference (delta) of thesignal on conventional T cells versus the one on Tregs (FIG. 10D). The2+1 and the 2+2 formats did not show an avidity driven selectivity foreffector T cells and are comparable in their binding to monospecificanti-LAG3 antibody.

12.3 Effect of PD-1 and LAG-3 Blockade on Treg Suppression of Granzyme Band IFN-g Release by Human CD4 T Cells Cocultured with IrradiatedAllogeneic PBMCs

It was further tested whether the differences in binding property of thebispecific antibody formats would provide any functional advantage toTconv over Tregs. In functional studies involving regulatory T cells(Treg)-suppression assays, PBMCs from the same donor where divided intwo samples: one was enriched in CD4 T cells and the other one in Tregsdefined as CD4⁺CD25^(high)CD127^(low) T cells via a microbead kit(Miltenyi Biotec). Once the two populations were purified, CD4 T cellswere labelled with 5 mM of Carboxy-Fluorescein-Succinimidyl Esther(CFSE) while Tregs were labelled with 5 mM Cell-Trace-Violet (CTV) to beable to distinguish them at the FACS later on.

Both CD4 T cells (10⁵) and Tregs (10⁵) were then co-cultured in a 96well plate at 1:1 ratio together with irradiated PBMCs (10⁵) from anunrelated donor in presence or absence of our anti-LAG3 antibodies (lead0414 and backup 0416) or competitor anti-LAG3 antibodies (BMS-986016 andhumanized BAP050) in combination with our anti-PD1 antibody at theconcentration of 10 mg/ml. As control to estimate the magnitude of thesuppression of CD4 T cell effector functions by Tregs, CD4 T cells (10⁵)were also co-cultured with irradiated PBMCs (10⁵) in the absence ofTregs.

Five days later we collected the cell-culture supernatants, used laterto measure IFNγ levels by ELISA (R&D systems), and left the cells at 37°C. for additional 5 hours in presence of Golgi Plug (Brefeldin A) andGolgi Stop (Monensin). The cells were then washed, stained on thesurface with anti-human CD4 antibody and the Live/Dead fixable dye Aqua(Invitrogen) before being fixed/permeabilized with Fix/Perm Buffer (BDBioscience). We performed intracellular staining for Granzyme B (BDBioscience) and IFNγ (eBioscience). Results are shown in FIG. 11.

Our PD1/LAG-3 bispecific antibody (0927) elicited Tconv escape fromregulatory T cell tight control as demonstrated by the secretion ofsignificantly higher amount of Granzyme B than Tconv in presence ofparental anti-PD1 antibody or Pembrolizumab alone (P<0.05) or in absenceof checkpoint inhibitors (P<0.001). Competitor anti-LAG3 antibodyBMS-986016 in combination with Nivolumab did not significantly rescueTconv effector functions from Treg suppression.

12.4 Effect of PD-1 and LAG-3 Blockade on Granzyme B and IFN-γ Secretionby CD4 T Cells from Melanoma Patient PBMCs after Recall with ImmunogenicMelanoma-Antigen Peptide Pools

It has been previously described that melanoma patient PBMCs containdetectable frequencies of tumor-antigen specific T cells. Therefore, forproof of concept purposes, the combination of anti-LAG-3 antibody (0414)plus anti-PD-1(0376) versus the derived bispecific antibody in 1+1(0927) format or anti-PD-1 alone were tested on melanoma patient PBMCsre-stimulated overnight with immunogenic melanoma associated antigenspeptide pools.

10⁵ to 10⁶ PBMCs from melanoma patients where incubated at roomtemperature in presence or absence of saturating concentrations (10μg/ml) of anti-PD-1 alone (0376), in combination with anti-LAG-3 (0414,10 μg/ml) antibody or as bispecific 1+1 format (0927, 20 μg/ml)antibody. T cells were then re-stimulated over-night with a pool ofimmunogenic tumor related antigens like MAGEA1, MAGEA3, MAGEA4,Melan-A/MART-1, NYESO-1. Melanocyte protein Pmel 17 gp100, Tyrosinase,Tyrosinase-related protein 2 in presence of protein transport inhibitorsGolgi Plug (Brefeldin A) and Golgi Stop (Monensin).

The cells were then washed, stained on the surface with anti-human CD4antibody and the Live/Dead fixable dye Aqua (Invitrogen) before beingfixed/permeabilized with Fix/Perm Buffer (BD Bioscience). Intracellularstaining was performed for Granzyme B (BD Bioscience) and IFN-γ(eBioscience).

Both the combination of anti-LAG-3 and anti-PD-1 antibodies (P<0.01 andP<0.001) and the bispecific antibody significantly (P<0.01 and P<0.0001)enhanced tumor-antigen specific T cell effector functions (i.e. GranzymeB and IFN-γ secretion) while PD-1 blockade alone did not show any effect(FIG. 12).

12.5 Effect of PD-1/LAG-3 Bispecific Antibodies on Cytotoxic Granzyme BRelease by Human CD4 T Cells Cocultured with a B Cell-Lymphoblatoid CellLine (ARH77)

We assessed the ability of our different bispecific antibody formats toinduce Granzyme B secretion by CD4 T cells, when co-cultured with thetumor cell line ARH77, in comparison to the combination of anti-PD-1 andanti-LAG-3 parental antibodies and to anti-PD1 antibodies used instandard of care.

In total 6 formats were tested, 3 generated from the combination ofanti-PD1(0376) and anti-LAG3 (hu 1256, chi 0414) antibodies and 3additional formats from anti-PD1 (0376) and anti-LAG-3 (hu1257, chi0416) antibodies.

As can be seen in FIG. 13, two bispecific formats, 1+1 (0927) and 2+2(8970) generated from anti-LAG3 (hu 1256, anti-LAG3 0414 as IgG1 PGLALA)and anti-PD1(0376) and as well as the combination of the parentalantibodies significantly enhanced Granzyme B secretion by CD4 T cellswhen compared to untreated CD4 T cells (P=0.0005, P=0.01 and P=0.0001respectively). The corresponding 2+1 format (8310) showed a similartrend, however it did not reach statistical significance (P=0.07).

Regarding the bispecific antibodies generated by combining anti-LAG-3(hu 1257, anti-LAG3 0416 as IgG1 PGLALA) with anti-PD1(0376), the 1+1format (0799) and 2+2 (8984) significantly increased the frequencies ofGranzyme B positive CD4 T cells when compared to untreated CD4 cells(P=0.0032 and P=0.0064 respectively).

Neither Nivolumab nor Pembrolizumab did significantly promote a higherGranzyme B secretion by CD4 T cells when compared to cells cultured inthe absence of checkpoint inhibitors. (FIG. 13).

TABLE 27 Effect of tested PD1-LAG3 bispecific antibodies on cytotoxicGranzyme B release Sample Effect PD1/LAG3 0927 (1 + 1) +++ PD1/LAG3 8970(2 + 2) + PD1/LAG3 8310 (1 + 2) +/− PD1/LAG3 0799 (1 + 1) ++ PD1/LAG38984 (2 + 2) ++ PD1/LAG3 8311 (1 + 2) +/− aLAG3(BMS986016) ++

Example 13 Potent Anti-Tumor Effect by Combination Therapy of PD1/LAG3Bispecific Antibodies and CEACAM5 CD3 TCB In Vivo

TCB molecules have been prepared according to the methods described inWO 2014/131712 A1 or WO 2016/079076 A1. The preparation of theanti-CEA/anti-CD3 bispecific antibody (CEA CD3 TCB or CEA TCB) used inthe experiments is described in Example 3 of WO 2014/131712 A1. CEA CD3TCB is a “2+1 IgG CrossFab” antibody and is comprised of two differentheavy chains and two different light chains. Point mutations in the CH3domain (“knobs into holes”) were introduced to promote the assembly ofthe two different heavy chains. Exchange of the VH and VL domains in theCD3 binding Fab were made in order to promote the correct assembly ofthe two different light chains. 2+1 means that the molecule has twoantigen binding domains specific for CEA and one antigen binding domainspecific for CD3. CEA CD3 TCB comprises the amino acid sequences of SEQID NO: 146, SEQ ID NO:147, SEQ ID NO: 148 and SEQ ID NO: 149. CEACAM5CD3 TCB has the same format, but comprises another CEA binder andcomprises point mutations in the CH and CL domains of the CD3 binder inorder to support correct pairing of the light chains. CEACAM5 CD TCBcomprises the amino acid sequences of SEQ ID NO:150, SEQ ID NO:151, SEQID NO:152 and SEQ ID NO:153.

a) Experimental Material and Methods

The PD1/LAG3 bispecific antibody 0927 was tested in a concentration of1.5 mg/kg or 3 mg/kg in combination with the human CEACAM5 CD3 TCB in ahuman pancreatic BXPC3 cancer model. BXPC3 cells were cograftedsubcutaneously with a mouse fibroblast cell line (3T3) in NSG humanizedmice.

Preparation of BXPC3 Cell Line:

BXPC3 cells (human pancreatic cancer cells) were originally obtainedfrom ECACC (European Collection of Cell Culture) and after expansiondeposited in the Glycart internal cell bank. BXPC3 cells were culturedin RPMI containing 10% FCS (PAA Laboratories, Austria), 1% Glutamax. Thecells were cultured at 37° C. in a water-saturated atmosphere at 5% CO₂.

Production of Fully Humanized Mice:

Female NSG mice, age 4-5 weeks at start of the experiment (JacksonLaboratory), were maintained under specific-pathogen-free condition withdaily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). The experimental study protocolwas reviewed and approved by local government (P 2011/128). Afterarrival, animals were maintained for one week to get accustomed to thenew environment and for observation. Continuous health monitoring wascarried out on a regular basis. The NSG mice were injected i.p. with 15mg/kg of Busulfan followed one day later by an i.v. injection of 1×105human hematopoietic stem cells isolated from cord blood. At week 14-16after stem cell injection mice were bled sublingual and blood wasanalyzed by flow cytometry for successful humanization. Efficientlyengrafted mice were randomized according to their human T cellfrequencies into the different treatment groups.

Efficacy Experiment:

Fully humanized HSC-NSG mice were challenged subcutaneously with 1×10⁶BXPC3 cells (human pancreatic carcinoma cell line, expressing CEACAM5)at day 0 in the presence of matrigel at 1:1 ratio. Tumors were measured2 to 3 times per week during the whole experiment by Caliper. At day 15mice were randomized for tumor size with an average tumor size of 250mm³ and a weekly scheduled therapy (vehicle (histidine buffer),anti-PD1(0376), Nivolumab, Pembrolizumab or anti PD1-LAG3 0927) startedand was given by intra-peritoneal injection in 400 μl max. Tumor growthwas measured 2-3 times weekly using a caliper and tumor volume wascalculated as followed:

T_(v):(W²/2)×L(W: Width,L: Length)

The study was terminated at day 47.

b) Results

The measurements of tumor volumes (mm³+/−SEM), over a period of 47 days,are shown as mean volume within the respective treatment group of micein FIG. 14. Treatment with CEACAM5-TCB only shows a disease progressionidentical to the untreated vehicle group Conversely, Nivolumab andPembrolizumab reduced the tumor growth, however, without reachingtumor-growth control. Surprisingly, PD1-LAG3 bispecific antibody 0927,at the concentration of 3 mg/Kg, fully suppressed tumor growth in alltreated animals showing synergism of the LAG-3 co-blockade in additionto PD-1.

In FIGS. 15A to 15F the measurements of tumor volumes (mm³+/−SEM), overa period of 47 days, are shown for each individual animal showing thehomogeneity of the anti-tumor response in each group.

The statistical significance was calculated by using the Dunnett'sMethod against the CEACAM5 CD3 TCB single treatment. To test forsignificant differences in group means for multiple comparisons, thestandard analysis of variance (ANOVA) is automatically produced, usingthe Dunnett's method. Dunnett's method tests whether means are differentfrom the mean of a control group.

The resulting TGI and TCR values are shown in Table 28 (TGI means tumorgrowth inhibition, TGI>100 means tumor regression and TGI=100 is definedas tumor stasis, TCR means treatment to control ratio, TCR=1 means noeffect and TCR=0 is defined as complete regression).

TABLE 28 Tumor growth inhibition (TGI) and Treatment to control ration(TCR) on day 46 Group (Day 46 reference CEACAM5-TCB TGI TCR p-valueCEACAM5 CD3 TCB 2.5 mg/kg + 93.06119 0.207878 0.0056 anti-PD1/LAG3 09271.5 mg/kg CEACAM5 CD3 TCB 2.5 mg/kg + 79.22326 0.006863 0.005anti-PD1/LAG3 0927 3 mg/kg CEACAM5 CD3 TCB 2.5 mg/kg + 32.9787 0.6685630.513 Nivolumab 1.5 mg/kg CEACAM5 CD3 TCB 2.5 mg/kg + 55.33328 0.4373980.07 Pembrolizumab 1.5 mg/kg

The comparison with the control is further shown as p-values usingDunnett's method.

The treatment with CEACAM5 CD3 TCB cannot control tumor growth in thecontext of pancreatic cancer. However, its combination with thebi-specific antibody anti-PD1/LAG3 0927, lead to a strong impact ontumor control in a dose specific manner. The statistical analysis showedthat the combination with anti-PD1/LAG3 0927, but not with the anti-PD1antibodies Nivolumab and Pembrolizumab, at both concentration resultedin statistical significant difference in control of tumor growth whencompared to single treatment, suggesting the superiority of thebi-specific anti-PD1/LAG3 antibody over inhibition of only PD1, bringingthe tumor growth to stasis.

1. A bispecific antibody comprising a first antigen binding domain thatspecifically binds to programmed cell death protein 1 (PD1) and a secondantigen binding domain that specifically binds to Lymphocyte activationgene-3 (LAG3), wherein said first antigen binding domain specificallybinding to PD1 comprises a VH domain comprising (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 1, (ii) HVR-H2 comprising theamino acid sequence of SEQ ID NO:2, and (iii) HVR-H3 comprising an aminoacid sequence of SEQ ID NO:3; and a VL domain comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:4; (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:5, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:6.
 2. The bispecificantibody of claim 1, wherein the bispecific antibody comprises a Fcdomain that is an IgG and wherein the Fc domain comprises one or moreamino acid substitution that reduces binding to an Fc receptor.
 3. Thebispecific antibody of claim 1, wherein the second antigen bindingdomain that specifically binds to LAG3 comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:14, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 15, and(iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO: 16; and aVL domain comprising (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 17, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 18, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO: 19; or (b) a VH domain comprising (i) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:22, (ii) HVR-H2 comprising the amino acidsequence of SEQ ID NO:23, and (iii) HVR-H3 comprising an amino acidsequence of SEQ ID NO:24; and a VL domain comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:25, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:26, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:27; or (c) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:30, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:31,and (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:32; anda VL domain comprising (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:33, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO:34, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:35; or (d) a VH domain comprising (i) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:38, (ii) HVR-H2 comprising the amino acidsequence of SEQ ID NO:39, and (iii) HVR-H3 comprising an amino acidsequence of SEQ ID NO:40; and a VL domain comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:41, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:42, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:43; or (e) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:46, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:47,and (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:48; anda VL domain comprising (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:49, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO:50, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:51.
 4. The bispecific antibody according to claim 1, wherein thefirst antigen-binding domain specifically binding to PD1 comprises (a) aVH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 8, or (b) a VHdomain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10, or (c) a VHdomain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 11, or (d) a VHdomain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 12, or (e) a VHdomain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO:
 13. 5. Thebispecific antibody according to claim 1, wherein the firstantigen-binding domain specifically binding to PD1 comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 9 and a VL domaincomprising the amino acid sequence of SEQ ID NO:
 10. 6. The bispecificantibody according to claim 3, wherein the second antigen-binding domainspecifically binding to LAG3 comprises (a) a VH domain comprising theamino acid sequence of SEQ ID NO: 20 and a VL domain comprising theamino acid sequence of SEQ ID NO: 21, or (b) a VH domain comprising theamino acid sequence of SEQ ID NO: 28 and a VL domain comprising theamino acid sequence of SEQ ID NO: 29, or (c) a VH domain comprising theamino acid sequence of SEQ ID NO: 36 and a VL domain comprising theamino acid sequence of SEQ ID NO: 37, or (d) a VH domain comprising theamino acid sequence of SEQ ID NO: 44 and a VL domain comprising theamino acid sequence of SEQ ID NO: 45, or (e) a VH domain comprising theamino acid sequence of SEQ ID NO: 52 and a VL domain comprising theamino acid sequence of SEQ ID NO:
 53. 7. The bispecific antibodyaccording to claim 3, wherein the second antigen-binding domainspecifically binding to LAG3 comprises (a) a VH domain comprising theamino acid sequence of SEQ ID NO: 54 and a VL domain comprising theamino acid sequence of SEQ ID NO: 55, or (b) a VH domain comprising theamino acid sequence of SEQ ID NO: 62 and a VL domain comprising theamino acid sequence of SEQ ID NO: 63, or (c) a VH domain comprising theamino acid sequence of SEQ ID NO: 64 and a VL domain comprising theamino acid sequence of SEQ ID NO: 65, or (d) a VH domain comprising theamino acid sequence of SEQ ID NO: 66 and a VL domain comprising theamino acid sequence of SEQ ID NO:
 67. 8. The bispecific antibodyaccording to claim 1, wherein the first antigen binding domainspecifically binding to PD1 comprises a VH domain comprising the aminoacid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acidsequence of SEQ ID NO: 10, and the second antigen binding domainspecifically binding to LAG3 comprises a VH domain comprising the aminoacid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acidsequence of SEQ ID NO: 21 or a VH domain comprising the amino acidsequence of SEQ ID NO: 52 and a VL domain comprising the amino acidsequence of SEQ ID NO:
 53. 9. The bispecific antibody according to claim1, wherein the first antigen binding domain specifically binding to PD1comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, andthe second antigen binding domain specifically binding to LAG3 comprisesa VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VLdomain comprising the amino acid sequence of SEQ ID NO:
 21. 10. Thebispecific antibody according to claim 1, wherein the first antigenbinding domain specifically binding to PD1 comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 9 and a VL domaincomprising the amino acid sequence of SEQ ID NO: 10, and the secondantigen binding domain specifically binding to LAG3 comprises a VHdomain comprising the amino acid sequence of SEQ ID NO: 56 and a VLdomain comprising the amino acid sequence of SEQ ID NO:
 57. 11. Thebispecific antibody of claim 1, wherein the bispecific antibodycomprises an Fc domain of human IgG1 subclass with the amino acidmutations L234A, L235A and P329G (numbering according to Kabat EUindex).
 12. The bispecific antibody of claim 1, wherein the bispecificantibody comprises an Fc domain comprising a modification promoting theassociation of the first and second subunit of the Fc domain.
 13. Thebispecific antibody of claim 12, wherein the first subunit of the Fcdomain comprises the amino acid substitutions S354C and T366W (numberingaccording to Kabat EU index) and the second subunit of the Fc domaincomprises the amino acid substitutions Y349C, T366S and Y407V (numberingaccording to Kabat EU index).
 14. The bispecific antibody of claim 1,wherein the bispecific antibody comprises an Fc domain, a first Fabfragment comprising the antigen binding domain that specifically bindsto PD1 and a second Fab fragment comprising the antigen binding domainthat specifically binds to LAG3.
 15. The bispecific antibody of claim14, wherein in one of the Fab fragments the variable domains VL and VHare replaced by each other so that the VH domain is part of the lightchain and the VL domain is part of the heavy chain.
 16. The bispecificantibody of claim 14, wherein in the first Fab fragment comprising theantigen binding domain that specifically binds to PD1 the variabledomains VL and VH are replaced by each other.
 17. The bispecificantibody of claim 1, wherein the bispecific antibody comprises a Fabfragment wherein in the constant domain CL the amino acid at position124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat EU Index), and in theconstant domain CH1 the amino acids at positions 147 and 213 aresubstituted independently by glutamic acid (E) or aspartic acid (D)(numbering according to Kabat EU index).
 18. The bispecific antibody ofclaim 1, wherein in the second Fab fragment comprising the antigenbinding domain that specifically binds to LAG3 the constant domain CLthe amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat EUIndex), and in the constant domain CH1 the amino acids at positions 147and 213 are substituted independently by glutamic acid (E) or asparticacid (D) (numbering according to Kabat EU index).
 19. The bispecificantibody of claim 1, comprising (a) a first heavy chain comprising anamino acid sequence with at least 95% sequence identity to the sequenceof SEQ ID NO: 96, a first light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 98, asecond heavy chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 97, and a second lightchain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO:99, or (b) a first heavy chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 96, a first light chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 98, a second heavy chain comprising an amino acid sequence withat least 95% sequence identity to the sequence of SEQ ID NO: 100, and asecond light chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 101, or (c) a firstheavy chain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 102, a first light chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 104, a second heavy chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 103, and a second light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 105,or (d) a first heavy chain comprising an amino acid sequence with atleast 95% sequence identity to the sequence of SEQ ID NO: 106, a firstlight chain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 107, a second heavy chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 103, and a second light chain comprising anamino acid sequence with at least 95% sequence identity to the sequenceof SEQ ID NO:
 105. 20. The bispecific antibody of claim 1, comprising afirst heavy chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 96, a first light chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 98, a second heavy chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 100, and a second light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO:101.21. The bispecific antibody of claim 14, wherein the bispecific antibodycomprises a second Fab fragment comprising the antigen binding domainthat specifically binds to LAG3 which is fused to the C-terminus of theFc domain.
 22. The bispecific antibody of claim 1, comprising a firstheavy chain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 96, a first light chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 98, a second heavy chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 144, and a second light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO:101.23. The bispecific antibody of claim 14, wherein the bispecific antibodycomprises a third Fab fragment comprising an antigen binding domain thatspecifically binds to LAG3.
 24. The bispecific antibody of claim 14,wherein the Fab fragment comprising the antigen binding domain thatspecifically binds to PD1 is fused via a peptide linker to theC-terminus of one of the heavy chains.
 25. The bispecific antibody ofclaim 1, comprising (a) a first heavy chain comprising an amino acidsequence with at least 95% sequence identity to the sequence of SEQ IDNO: 118, a first light chain comprising an amino acid sequence with atleast 95% sequence identity to the sequence of SEQ ID NO: 115, a secondheavy chain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 119, and two second light chainscomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 101, or (b) a first heavy chain comprising anamino acid sequence with at least 95% sequence identity to the sequenceof SEQ ID NO: 120, a first light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 115, asecond heavy chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 121, and two secondlight chains comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO:99, or (c) a first heavychain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 122, a first light chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 115, a second heavy chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 103, and two second light chains comprising an amino acidsequence with at least 95% sequence identity to the sequence of SEQ IDNO:
 105. 26. The bispecific antibody of claim 14, wherein one of the Fabfragments comprising the antigen binding domain that specifically bindsto LAG3 is fused via a peptide linker to the C-terminus of one of theheavy chains.
 27. The bispecific antibody of claim 1, comprising (a) afirst heavy chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 96, a first light chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 98, a second heavy chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 145, and two second light chains comprising an amino acidsequence with at least 95% sequence identity to the sequence of SEQ IDNO:
 101. 28. The bispecific antibody of claim 23, wherein the bispecificantibody comprises a fourth Fab fragment comprising an antigen bindingdomain that specifically binds to PD1.
 29. The bispecific antibody ofclaim 28, wherein the two Fab fragments comprising each an antigenbinding domain that specifically binds to PD1 are identical.
 30. Thebispecific antibody of claim 28, wherein the two Fab fragmentscomprising each an antigen binding domain that specifically binds to PD1are each fused via a peptide linker to the C-terminus to one of theheavy chains, respectively.
 31. The bispecific antibody of claim 1,comprising (a) two heavy chains comprising each an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 114,two first light chains comprising each an amino acid sequence with atleast 95% sequence identity to the sequence of SEQ ID NO: 115, and twosecond light chains comprising each an amino acid sequence with at least95% sequence identity to the sequence of SEQ ID NO: 101, or (b) twoheavy chains comprising each an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 116, two first lightchains comprising each an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 115, and two second light chainscomprising each an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO:99, or (c) two heavy chainscomprising each an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 117, two first light chainscomprising each an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 115, and two second light chainscomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO:
 105. 32. The bispecific antibody of claim 1,wherein the bispecific antibody comprises an Fc domain, two Fabfragments comprising each an antigen binding domain that specificallybinds to LAG3 and a single chain Fab (scFab) comprising the antigenbinding domain that specifically binds to PD1.
 33. The bispecificantibody of claim 32, wherein the scFab comprising an antigen bindingdomain that specifically binds to PD1 is fused via a peptide linker tothe C-terminus to one of the heavy chains.
 34. The bispecific antibodyof claim 1, comprising (a) a first heavy chain comprising an amino acidsequence with at least 95% sequence identity to the sequence of SEQ IDNO: 123, a second heavy chain comprising an amino acid sequence with atleast 95% sequence identity to the sequence of SEQ ID NO: 119, and twolight chains comprising each an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 101, or (b) a firstheavy chain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 124, a second heavy chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 121, and two light chains comprising each anamino acid sequence with at least 95% sequence identity to the sequenceof SEQ ID NO:99, or (c) a first heavy chain comprising an amino acidsequence with at least 95% sequence identity to the sequence of SEQ IDNO: 125, a second heavy chain comprising an amino acid sequence with atleast 95% sequence identity to the sequence of SEQ ID NO: 103, and asecond light chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO:
 105. 35. The bispecificantibody of claim 1, wherein the bispecific antibody comprises an Fcdomain, two Fab fragments comprising each an antigen binding domain thatspecifically binds to LAG3 and a VH and VL domain comprising the antigenbinding domain that specifically binds to PD1.
 36. The bispecificantibody of claim 1, wherein the VH domain of the antigen binding domainthat specifically binds to PD1 is fused via a peptide linker to theC-terminus of one of the heavy chains and the VL domain of the antigenbinding domain that specifically binds to PD1 is fused via a peptidelinker to the C-terminus of the other one of the heavy chains.
 37. Thebispecific antibody of claim 1, comprising a first heavy chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 126, a second heavy chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 127, and two light chains comprising each an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 109.38. A polynucleotide encoding the bispecific antibody of claim
 1. 39. Aprokaryotic or eukaryotic host cell comprising the polynucleotideaccording to claim
 38. 40. A method of producing the bispecific antibodyaccording to claim 1, comprising culturing the host cell of claim 39under conditions suitable for the expression of the bispecific antibodyand recovering the bispecific antibody from the culture.
 41. Apharmaceutical composition comprising the bispecific antibody accordingto claim 1 and at least one pharmaceutically acceptable excipient. 42.(canceled)
 43. (canceled)
 44. (canceled)
 45. (canceled)
 46. (canceled)47. (canceled)
 48. (canceled)
 49. A method of treating an individualhaving cancer or a chronic viral infection comprising administering tothe individual an effective amount of the bispecific antibody ofclaim
 1. 50. A method of inhibiting the growth of tumor cells in anindividual comprising administering to the individual an effectiveamount of the bispecific antibody according to claim 1 to inhibit thegrowth of the tumor cells.